EA044919B1 - METHODS FOR REDUCING OR ELIMINATING PROTEIN MODIFICATION AND DECOMPOSITION CAUSED BY EXPOSURE TO UV RADIATION - Google Patents

Description

Field of technology

The present invention relates generally to compounds and methods for protecting protein-based molecules from degradation and modification during operations involving exposure of said protein-based molecules to UV radiation, particularly in viral inactivation methods that involve light in the UVC wavelength range waves, and also allows for enhanced UVC irradiation without damaging proteins.

State of the art

Viral contamination of cell media and supernatants poses a challenge to biopharmaceutical manufacturers worldwide. Various methods have been used to inactivate and/or remove viral particles, large or small, DNA or RNA, enveloped or naked, from cellular supernatants. Examples of such methods include 20 nm filtration technology, anion exchange membrane chromatography, low pH incubation and depth filtration technology.

In addition to the methods described above, treatment of protein-containing solutions with ultraviolet radiation was also used to inactivate viruses. However, to achieve effective inactivation of viruses, a sufficient dose of UV radiation in the UVC range must be supplied to the solution. In some cases, the required level of exposure to UVC radiation may cause undesirable modification and/or degradation of the protein in solution. For example, in some cases, reactive substances may form in solution, which can lead to indirect oxidation or modification of proteins in solution; Other mechanisms of indirect modification due to exposure to UVC are also possible. See, for example, Cabiscol, et al., (2010) Int. Microbiol 3:315; Bandyopadhyay et al. (1999) Curr. Sci. 77:658-666; Schoneich, (2005) Biochim Biophys Acta 1703:111-19; Stadtman et al., (2003) Antioxid. Redox. Signal 5:577-82; Stadtman, (1993) Ann. Rev. Biochem. 62:797-821 and Dean et al., (1997) Biochem. J. 324:1-18.

The present invention is directed to solving these and other problems by providing methods for reducing the oxidation, modification and degradation of protein in a solution exposed to UV radiation, and in particular UVC radiation. Exposure to UVC may be, as described herein, a component of a viral inactivation procedure, and the protein in solution may be any type of protein, for example a protein such as an antigen binding protein (e.g., any one or more of (i) an antigen binding protein containing that - one or more of a monoclonal antibody, human antibody, humanized antibody, chimeric antibody, recombinant antibody, single chain antibody, diabody, tribody, tetrabody, Fab fragment, F(ab') ₂ fragment, IgD antibody, IgE antibody , IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies or IgG4 antibodies and fragments thereof, (ii) Fc domain; (iii) peptide; (iv) fusion protein with Fc fragment and (v) therapeutic protein).

The essence of the invention

In one aspect, a method is provided for inactivating a virus in a sample containing a protein component. According to one embodiment, the method includes (a) providing a sample containing a protein component, the sample being known or suspected to contain a virus; (b) determining the target dose of UV radiation, under the influence of which the virus is inactivated; (c) adding a preservative to the sample to obtain a stabilized mixture; (d) exposing said stabilized mixture to UV radiation provided by a source operating at a selected power level and wavelength for a selected period of time; (e) assessing the level of exposure of the stabilized mixture to UVC; and (f) modulating any one or more of the wavelength, power of the UV source, and time of exposure to UV radiation if, according to such assessment, the stabilized mixture has not received the target dose UV radiation.

According to one embodiment, the specified sample contains a pool obtained on a chromatographic column; in certain embodiments, the pool may include any one or more of a Protein A column-derived protein component-containing eluent pool, a Protein G column-derived protein component-containing column eluent pool, and a hydrophobic interaction chromatography (GC) column eluent pool. a pool containing a protein component, a size exclusion chromatography (SEC) column pool containing a protein component, an ion exchange chromatography (IEC) column pool containing a protein component, and a hydroxyapatite column pool containing a protein component. According to another embodiment, said sample comprises an effluent from a chromatography column; in specific embodiments, said effluent stream may include any one or more of a protein A column effluent containing a protein component, a protein G column effluent containing a protein component, a hydrophobic interaction chromatography (GC) column effluent containing a protein component, a size exclusion chromatography (SEC) column effluent containing a protein component, an ion exchange chromatography (IEC) column effluent containing a protein component, and a hydroxyapatite column effluent containing a protein component.

In other embodiments, the protein component may comprise any one or more of (i) an antigen binding protein comprising any one or more of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a single chain antibody, a diabody, tribody, tetrabody, Fab fragment, F(ab')2 fragment, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody or IgG4 antibody, and fragments thereof, (ii) Fc domain; (iii) a peptide; (iv) a fusion protein with an Fc fragment; and (v) a therapeutic protein.

In further embodiments, the virus includes any one or more of a dsDNA virus, a ssDNA virus, a dsRNA virus, and a ssRNA virus; in specific embodiments, the virus may include a virus from one or more of the adenoviridae, asfarviridae, herpesviridae, iridoviridae, papillomaviridae, polyomaviridae, poxviridae, circoviridae, hepadnaviridae, parvoviridae, birnaviridae, reoviridae, arenaviridae, vornaviridae, bunyaviridae, deltaviridae, filo virus families viridae, orthomyxoviridae , paramyxoviridae, rhabdoviridae, arterioviridae, astroviridae, caliciviridae, coronaviridae, flaviviridae, hepatitis E-like viruses, nodaviridae, picornaviridae, togaviridae and tertroviridae.

In specific embodiments, the virus is a parvovirus MVM (Minor Mouse Virus), a murine leukemia retrovirus MuLV, or a sheep bunyavirus CVV.

In another embodiment, the protectant is added to the sample at a concentration ratio of greater than 1 part protectant to 200 parts protein; in other embodiments, said protective agent may contain any one or more of tyrosine, tryptophan, methionine, pyridoxine, and riboflavin. In specific embodiments, said protective agent comprises any one of (i) tyrosine; (ii) tryptophan and (iii) tyrosine and tryptophan.

In further embodiments, the UV wavelength is in the range of about 200 nm to about 280 nm; in specific embodiments, the UV wavelength is approximately 254 nm. The target dose, in various embodiments, may be any one or more of about 1 mJ/cm ² , about 10 mJ/cm ² , about 25 mJ/cm ² , about 50 mJ/cm ² , about 75 mJ /cm ² , approximately 100 mJ/cm ² , approximately 125 mJ/cm ² , approximately 200 mJ/cm 2 , approximately 250 mJ/cm ² , approximately 300 mJ/cm ² , approximately 350 mJ/ ^{cm 2 ,} approximately 400 mJ/ cm ² , approximately 450 mJ/cm ² , approximately 500 mJ/cm ² , approximately 600 mJ/cm ² , approximately 700 mJ/cm ² , approximately 800 mJ/cm ² , approximately 900 mJ/cm ² , approximately 1000 mJ/cm ² and more than approximately 1000 mJ/ ^cm2 .

In some embodiments, the method provides a log reduction in virus (LRV) greater than or equal to about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, or greater than about 6.5.

According to another embodiment, the method is automated; In yet another embodiment, the method is implemented as a step in a protein purification procedure.

In another aspect, a method is provided for reducing protein degradation or modification caused by the presence of reactive substances generated by UV exposure. In one embodiment, the method comprises (a) providing a sample containing a protein component known or suspected to be degraded or modified in the presence of reactive substances; (b) determining the target dose of UV radiation; (c) adding a preservative to the sample to obtain a stabilized mixture; (d) exposing said stabilized mixture to UV radiation provided by a source operating at a selected power level and wavelength for a selected period of time; (e) assessing the level of exposure of the stabilized mixture to UVC; and (f) modulating any one or more of the wavelength, power of the UV source, and time of exposure to UV radiation if, according to such assessment, the stabilized mixture has not received the target dose UV irradiation.

According to one embodiment, the specified sample contains a pool obtained on a chromatographic column; In certain embodiments, said pool may include any one or more of a Protein A column-derived protein component-containing eluent pool, a Protein G column-derived protein component-containing column eluent pool, and a hydrophobic interaction chromatography (GC) column eluent pool. a pool containing a protein component, a size exclusion chromatography (SEC) column pool containing a protein component, an ion exchange chromatography (IEC) column pool containing a protein component, and a hydroxyapatite column pool containing a protein component. According to

- 2 044919 In another embodiment, said sample contains an effluent from a chromatographic column; in certain embodiments, said effluent stream may comprise any one or more of a protein A column effluent containing a protein component, a protein G column effluent containing a protein component, a hydrophobic interaction chromatography (GC) column effluent containing a protein component, a size exclusion chromatography (SEC) column effluent containing a protein component, an ion exchange chromatography (IEC) column effluent containing a protein component, and a hydroxyapatite column effluent containing a protein component.

In other embodiments, the protein component may comprise any one or more of (i) an antigen binding protein comprising any one or more of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a single chain antibody, a diabody, tribody, tetrabody, Fab fragment, F(ab') ₂ fragment, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody or IgG4 antibody, and fragments thereof, (ii ) Fc domain; (iii) a peptide; (iv) a fusion protein with an Fc fragment; and (v) a therapeutic protein.

In yet another embodiment, the protectant is added to the sample at a concentration ratio of greater than 1 part protectant to 200 parts protein, and in other embodiments, the protectant may contain any one or more of tyrosine, tryptophan, methionine, pyridoxine and riboflavin. In specific embodiments, said protective agent comprises any one of (i) tyrosine; (ii) tryptophan and (iii) tyrosine and tryptophan.

In further embodiments, the UV wavelength is in the range of about 200 nm to about 280 nm; in specific embodiments, the UV wavelength is approximately 254 nm. The target dose, in various embodiments, may be any one or more of about 1 mJ/cm ² , about 10 mJ/cm ² , about 25 mJ/cm ² , about 50 mJ/cm ² , about 75 mJ /cm ² , approximately 100 mJ/cm ² , approximately 125 mJ/cm ² , approximately 200 mJ/cm 2 , approximately 250 mJ/cm ² , approximately 300 mJ/cm ² , approximately 350 mJ/ ^{cm 2 ,} approximately 400 mJ/ cm ² , approximately 450 mJ/cm ² , approximately 500 mJ/cm ² , approximately 600 mJ/cm ² , approximately 700 mJ/cm ² , approximately 800 mJ/cm ² , approximately 900 mJ/cm ² , approximately 1000 mJ/cm ² and more than approximately 1000 mJ/ ^cm2 .

In some embodiments, the method provides a log reduction in virus (LRV) greater than or equal to about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, or more than about 6.5.

In another embodiment, the method is automated, and in another embodiment, the method is implemented as a step in a protein purification procedure.

In another aspect, a method is provided for reducing the oxidation of methionine residues, tryptophan residues, or both methionine residues and tryptophan residues in a protein exposed to UV radiation. According to one embodiment, the method includes (a) providing a sample containing a protein component containing a methionine residue, a tryptophan residue, or both a methionine residue and a tryptophan residue; (b) determining the target dose of UV radiation; (c) adding a preservative to the sample to obtain a stabilized mixture; (d) exposing said stabilized mixture to UV radiation provided by a source operating at a selected power level and wavelength for a selected period of time; (e) assessing the level of exposure of the stabilized mixture to UVC; and (f) modulating any one or more of the wavelength, power of the UV source, and time of exposure to UV radiation if, according to such assessment, the stabilized mixture has not received the target dose UV irradiation.

According to one embodiment, the specified sample contains a pool obtained on a chromatographic column; In certain embodiments, said pool may include any one or more of a Protein A column-derived protein component-containing eluent pool, a Protein G column-derived protein component-containing column eluent pool, and a hydrophobic interaction chromatography (GC) column eluent pool. a pool containing a protein component, a size exclusion chromatography (SEC) column pool containing a protein component, an ion exchange chromatography (IEC) column pool containing a protein component, and a hydroxyapatite column pool containing a protein component. According to another embodiment, said sample comprises an effluent from a chromatography column; in certain embodiments, said effluent stream may comprise any one or more of a protein A column effluent containing a protein component, a protein G column effluent containing a protein component, a hydrophobic interaction chromatography (GC) column effluent containing a protein component, a size exclusion chromatography (SEC) column effluent containing a protein component, an ion exchange chromatography (IEC) column effluent containing a protein component, and a hydroxyapatite column effluent containing a protein component.

In other embodiments, the protein component may comprise any one or more of (i) an antigen binding protein comprising any one or more of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a single chain antibody, a diabody, a tribody , tetrabody, Fab fragment, F(ab') ₂ fragment, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody or IgG4 antibody, and fragments thereof, (ii) Fc -domain; (iii) a peptide; (iv) a fusion protein with an Fc fragment; and (v) a therapeutic protein.

In another embodiment, the protectant is added to the sample at a concentration ratio of greater than 1 part protectant to 200 parts protein; in other embodiments, the protectant may contain any one or more of tyrosine, tryptophan, methionine, pyridoxine, and riboflavin. In specific embodiments, the protectant comprises any one of (i) tyrosine; (ii) tryptophan and (iii) tyrosine and tryptophan.

In further embodiments, the UV wavelength is in the range of about 200 nm to about 280 nm; in specific embodiments, the UV wavelength is approximately 254 nm. Said target dose, in various embodiments, may be any one or more of about 1 mJ/ ^cm2 , about 10 mJ/ ^cm2 , about 25 mJ/ ^cm2 , about 50 mJ/ ^cm2 , about 75 mJ/ ^cm2 , approximately 100 mJ/ ^cm2 , approximately 125 mJ/ ^cm2 , approximately 200 mJ/cm2, approximately 250 mJ/ ^cm2 , approximately 300 mJ/ ^cm2 , approximately 350 mJ/ ^{cm2 ,} approximately 400 mJ /cm ² , approximately 450 mJ/cm ² , approximately 500 mJ/cm ² , approximately 600 mJ/cm ² , approximately 700 mJ/cm ² , approximately 800 mJ/cm ² , approximately 900 mJ/cm ² , approximately 1000 mJ/ cm ² and more than approximately 1000 mJ/cm ² .

In some embodiments, the method provides a log reduction in virus (LRV) greater than or equal to about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, or greater than about 6.5.

According to another embodiment, the method is automated; According to another embodiment, the method is implemented as a step in a protein purification procedure.

Brief description of the figures

Fig. 1 is a plot of the percentage changes in the main peak in three different monoclonal antibody samples (Mab X, Mab Y, Mab Z) as a function of UVC exposure monitored at three different pH levels (pH 4.3, 5.0 and 7.0 ) using size exclusion HPLC (SEC-HPLC); along the y-axis the changes in the value are plotted as a percentage relative to the dose of 0 mJ arriving at the surface of the solution, and along the x-axis the calculated dose arriving (mJ/cm ² ) at the surface of the solution is plotted. All samples were filtered virus-inactivated pools (FVIP).

Fig. 2 is a plot of major peak, acid peak, and base peak purity for two different monoclonal antibody samples (Mab X, Mab Z) as a function of UVC exposure monitored at three different pH levels (pH 4.3, 5.0, and 7. 0) by COX-HPLC analysis; The % of distribution of the measured substance relative to the dose of 0 mJ/cm ² arriving at the surface of the solution is plotted along the y-axis, and the calculated dose (mJ/cm ² ) arriving at the surface of the solution is plotted along the x-axis. All samples were filtered virus-inactivated pools (FVIP).

Fig. 3 is a plot of the % change in the main peak for three different monoclonal antibody samples (Mab X, Mab Y, Mab Z) as a function of UV exposure monitored at three different conductivity levels (low (30mM HOAc), standard (100mM HOAc) , and high (300 mM HOAc)) by SEC-HPLC analysis; The % change in value relative to the dose of 0 mJ/cm ² arriving at the surface of the solution is plotted along the y-axis, and the calculated dose (mJ/cm ² ) arriving at the surface of the solution is plotted along the x-axis.

Fig. 4 is a graph of the main peak, acid peak, and base peak purity of two different monoclonal antibody samples (Mab X, Mab Z) as a function of UVC exposure monitored at three different conductivity levels (low (30 mM HOAc), standard (100 mM HOAc) , and high (300 mM HOAc)) by KOX-HPLC analysis; along the y-axis the distribution of the measured substance is plotted in % relative to the dose of 0 mJ/ ^cm2 arriving at the surface of the solution; The calculated dose (mJ/ ^cm2 ) arriving at the surface of the solution is plotted along the x-axis. All samples were filtered virus-inactivated pools (FVIP).

In fig. 5 is a graph of the main peak concentration of a monoclonal antibody (Mab X) as a function of UVC exposure monitored at three different concentrations (low (6 g/L), standard (12 g/L), high (24 g/L)) using SEC-HPLC analysis; along the y-axis the % change in value relative to the dose of 0 mJ/ ^cm2 arriving at the surface of the solution is plotted, and along the x-axis the calculated dose received (mJ/ ^cm2 ) is plotted, taking into account the absorption capacity in relation to the UV components of the solution.

In fig. 6 shows a plot of purity and three major peak contours for a monoclonal antibody (Mab X) as a function of UVC exposure as monitored using SEC-HPLC analysis. The graph shows two Mab X peptide maps: Assay 1 with UV irradiation and Assay 2 with UV irradiation. The % change in value relative to the dose of 0 mJ/cm ² arriving at the solution surface is plotted along the y-axis, and the calculated dose (mJ/cm ² ) arriving at the solution surface is plotted along the x-axis.

Fig. 7 is a peptide map showing the oxidation effects observed on amino acid residues of a monoclonal antibody (Mab X) after exposure to a dose of 10,000 mJ/cm ² UVC applied to the surface of the solution. Oxidation of methionine residues at positions 425 and 249 was observed upon exposure to this extreme dose of UVC.

Fig. 8 is a table of summarized data on purity and activity trends for the monoclonal antibody (Mab X) as a function of UVC exposure at doses of 0, 150, 375 and/or 1000 mJ/cm ² . The results of peptide mapping indicate the oxidation of specific methionine residues on the protein, the content of which increases with increasing dose. Mab X is designated as Control; Tyrosine is designated as Additive 1.

Fig. 9 is a plot of monoclonal antibody (Mab X) major peak purity as a function of UVC exposure in the presence and absence of protective agent as monitored by SEC-HPLC analysis; % of the main peak is plotted on the y-axis as an indicator of the distribution of the measured substance; The calculated dose arriving at the surface of the solution (mJ/cm ² ) is plotted along the x axis. The graph shows three Mab X peptide maps: for Assay 1 with UV irradiation and Assay 2 with UV irradiation, for duplicate assays without treatment, and for the assay with Additive #2 under UV irradiation and tryptophan protection.

Fig. 10 is a graph of xmuLV inactivation as a function of UVC exposure in the presence and absence of protectant and monoclonal antibody (Mab Y). The y-axis represents the relative log reduction relative to a given known viral load (log reduction value (LRV)). The calculated dose received is plotted along the x-axis, taking into account the absorption capacity of the solution components in relation to UV (mJ/ ^cm2 ). The protein concentration in the Control sample (Mab Y) is 2 g/L and 30 g/L, the protein concentration in the Additive 1 sample (Mab Y + tyrosine) is 2 g/L and 30 g/L.

In fig. 11a and 11b present tables of UVC doses required to inactivate DNA viruses (Fig. 11a) and RNA viruses (Fig. 11b).

In fig. 12 is a table showing the predicted log reduction values (LRV) as a function of the UV dose required to inactivate various viruses.

Fig. 13 is a schematic representation of multiple UVC sources operating in parallel to provide processing of large volumes of stabilized sample.

Fig. 14 is a plot of monoclonal antibody (Mab X) major peak purity as a function of UVC exposure in the presence of various protectants, including tyrosine (TYR), tryptophan (TRP), phenylalanine (PHE), folic acid, methionine (MET), and histidine ( HIS) as monitored by SEC-HPLC analysis; % of the main peak is plotted on the y-axis as an indicator of the distribution of the measured substance; The calculated dose arriving at the surface of the solution is plotted along the x-axis. The molar ratio of amino acid to Mab X is 20:1. Mab X control is also introduced.

Fig. 15 is a plot of monoclonal antibody (Mab X) major peak purity as a function of UVC exposure in the presence of various protectants, including tyrosine (TYR), tryptophan (TRP), phenylalanine (PHE), folic acid, methionine (MET), and histidine ( HIS) as monitored by EC-HPLC analysis and is presented as a percentage change in initial purity; the y-axis represents the normalized percentage change in the purity of the main peak; The calculated dose arriving at the surface of the solution is plotted along the x-axis. The molar ratio of amino acid to Mab X is 20:1. Mab X control is also introduced.

Detailed Description of the Invention

The invention described herein provides methods for treating solutions containing proteins with radiation corresponding to the C region of the UV spectrum (UVC, approximately 254 nm). In particular, the invention described herein provides a method for treating solutions containing proteins with radiation corresponding to the C region of the UV spectrum (UVC, approximately 254 nm) in the presence of chemicals that stabilize the specified protein or minimize protein modification, for example, oxidation residues such as methionine and tryptophan residues.

The viral inactivation methods proposed herein aim to achieve a precise balance in which protein damage, modification or deterioration is minimized while maximizing the ability to inactivate viral and virus-related particles. This application proposes various additives for solutions capable of protecting proteins in solution from indirect modification by substances formed during treatment of the solution with radiation in the UV range. Data were collected for a number of polypeptide molecules, particularly antigen-binding proteins (eg, monoclonal antibody molecules), to determine the effects of UVC on protein modification. A relationship has been established between the dose level (mJ of UVC transferred/cm ² ) and the level of protein modification, as assessed by EC-HPLC (for analysis of aggregation and dimerization), COX-HPLC (for analysis of charge changes), peptide mapping (for analysis of molecular modification , oxidation and other effects) and biological activity (for analysis of molecular effectiveness).

Accordingly, in one aspect, the present invention relates to a method for inactivating a virus in a sample containing a protein component. This method involves the use of UV radiation to inactivate the virus. One of the beneficial aspects of the described method is that it uses a protectant that can minimize the possibility of degradation or modification of the protein component of the sample. The method may include a feedback component in which the sample containing the protective agent is monitored to ensure that the sample is receiving a dose of UV radiation sufficient to inactivate the virus while minimizing exposure of the protein component to damaging doses of UV radiation. protein. This method allows for effective destruction of viruses in the sample, while at the same time minimizing protein degradation, and can be useful when working on a large scale, both in terms of sample volume and parallelism of procedures, in particular as this method allows scaling up from the laboratory from scales involving crops on the order of a few liters to production scales involving crops in the thousands of liters.

According to another aspect, the present invention relates to a method of reducing the degradation or modification of a protein in the presence of reactive substances, such as reactive substances that can be formed when a solution is exposed to UV radiation. It has been noted that proteins can be degraded or modified by prolonged exposure to UV radiation. By including a protectant in a sample containing a protein component, protein damage can be reduced or eliminated. The method may include a feedback component where the sample containing the protective agent is monitored to ensure that the sample is receiving a dose of UV radiation sufficient to inactivate the virus while minimizing exposure of the protein component to doses of UV radiation that could damage the protein . This method effectively protects the protein component of the sample from degradation or modification of the protein and can be useful when working on a large scale, both in terms of sample volume and parallelism of procedures, in particular since this method allows scaling up from laboratory scale, including culture volumes on the order of a few liters, to production scales involving crops amounting to thousands of liters.

In yet another aspect, the present invention provides a method for reducing the oxidation of methionine residues, tryptophan residues, or both methionine residues and tryptophan residues in a protein exposed to UV radiation. It has been noted that tryptophan and methionine can be oxidized by prolonged exposure to UV radiation. By including a protectant in a sample containing a protein component, such oxidation can be reduced or eliminated. The method may include a feedback component in which the sample containing the protective agent is monitored to ensure that the sample is receiving a dose of UV radiation sufficient to inactivate the virus while minimizing exposure of the protein component to damaging doses of UV radiation. protein. This method effectively protects the protein component of the sample from degradation or modification of the protein and can be useful when working on a large scale, both in terms of sample volume and parallelism of procedures, in particular since this method allows scaling up from laboratory scale, including culture volumes on the order of a few liters, to production scales involving crops amounting to thousands of liters.

One of the advantages of the methods described is that they can be implemented at a variety of scales, from laboratory scale (typically in millimeters or liters), pilot plant scale (typically hundreds of liters) or industrial scale (typically thousand liters). In addition, this method can also be implemented multiple times, in parallel or sequentially. Accordingly, this method can be easily automated. The use of the described methods on a large scale may be advisable, in particular, in the production of biomolecules.

I. Definitions.

As used herein, the singular terms mean one or more unless specifically stated otherwise.

As used herein, the term antigen refers to a molecule or part of a molecule,

- 6 044919 capable of binding to a selective binding agent, such as an antigen binding protein (including, for example, an antibody or an immunologically functional fragment thereof), and may also be suitable for use in an animal to produce antibodies capable of binding said antigen. An antigen may contain one or more epitopes that are capable of interacting with various antigen-binding proteins, such as antibodies.

As used herein, the term antigen binding protein refers to a protein comprising a portion that binds to an antigen or target and, optionally, a scaffold or framework portion that allows said antigen binding portion to adopt a conformation that facilitates the binding of said antigen binding protein to an antigen. Examples of antigen binding proteins include monoclonal antibody; human antibody; humanized antibody; chimeric antibody; recombinant antibody; single chain antibody; diabody; triatelo; tetrabody; domain antibody; Fab fragment; F(ab') ₂ - fragment; IgD antibody; IgE antibody; IgM antibody; IgG1 antibody; IgG2 antibody; IgG3 antibody or IgG4 antibody; and their fragments. The antigen binding protein may comprise, for example, an alternative protein scaffold or an artificial scaffold with grafted CDR regions or derivative CDR regions. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing modifications introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein, as well as fully synthetic scaffolds containing, for example, a biocompatible polymer. See, for example, Korndorfer et al., (2003) Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129; Roque et al., (2004) Biotechnol. Prog. 20:639-654. In addition, the scaffold can be formed by peptide antibody mimetics (PAMs); Scaffolds can also be based on antibody mimetics with fibronectin components.

The antigen binding protein may have, for example, the structure of a naturally occurring immunoglobulin. Immunoglobulin is a tetrameric molecule. In naturally occurring immunoglobulin, each tetramer consists of two identical pairs of polypeptide chains, each pair containing one light chain (approximately 25 kDa) and one heavy chain (approximately 50-70 kDa). The amino-terminal part of each chain includes a variable region of approximately 100-110 or more amino acids in length, which is primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are divided into kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotype IgM, IgD, IgG, IgA, and IgE, respectively. The variable and constant regions of the light and heavy chains are connected by a J region of approximately 12 or more amino acids, while the heavy chain also includes a D region of approximately 10 additional amino acids. See generally Fundamental Immunology ^2nd ed. Ch. 7 (Paul, W., ed., Raven Press, NY (1989)), incorporated herein by reference in its entirety and for all purposes. The variable regions of each light/heavy chain pair form the antibody binding site, so that an intact immunoglobulin contains two binding sites.

Naturally occurring immunoglobulin chains exhibit the same general structure with relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity determining regions, or CDRs. From the N-terminus towards the C-terminus, both light and heavy chains contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. Numbering of amino acids in each of the domains can be carried out in accordance with the definitions of Kabat et al., (1991) Sequences of Proteins of Immunological Interest, ^5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242. Although the CDRs disclosed herein are described using Kabat nomenclature, if desired, they may also be redefined according to an alternative nomenclature scheme, such as Chothia (see Chothia & Lesk, (1987) J. Mol. Biol. 196:901 -917; Chothia et al., (1989) Nature 342:878-883 or Honegger & Pluckthun, (2001) J. Mol. Biol. 309:657-670).

As used herein, the term antibody refers to an intact immunoglobulin or an antigen-binding portion thereof that competes with the intact antibody for specific binding, unless otherwise noted. Antigen-binding sites can be generated using recombinant DNA techniques, or by enzymatic or chemical digestion of intact antibodies. Antigen binding regions include, but are not limited to, Fab, Fab', F(ab') ₂ , Fv, domain antibodies (dAbs), fragments including complementarity determining regions (CDRs), single chain antibodies (scFv), chimeric antibodies, diabodies, tribodies , tetrabodies and polypeptides that contain at least a portion of an immunoglobulin sufficient to impart specific antigen-binding ability to the polypeptide.

The Fab fragment is a monovalent fragment containing V _L , V _H , C _L and CH1 domains; The F(ab') ₂ fragment is a bivalent fragment containing two Fab fragments connected by a disulfide bridge at the hinge region; The Fd fragment contains VH and CH1 domains; The Fv fragment contains the V _L and VH domains of a single arm antibody; and the dAb fragment contains a VH domain, V _L

- 7 044919 domain or antigen binding fragment of the VH domain or V _L domain (US patent No. 6846634 and

6696245; Published US Patent Applications Nos. 05/0202512, 04/0202995, 04/0038291, 04/0009507,

03/0039958, Ward et al., Nature 341:544-546 (1989)).

A single chain antibody (scFv) is an antibody in which the V _L and V H regions are connected by a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, wherein the linker is of sufficient length to allow the protein chain to fold to form a monovalent antigen binding site (See, for example, Bird et al., (1988) Science 242:423-26 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-83). Diabodies are bivalent antibodies containing two polypeptide chains, with each polypeptide chain containing VH and _VL domains connected by a linker too short to allow pairing between two domains of the same chain, thereby allowing pairing of each domain with a complementary domain of another polypeptide chain (see, for example, Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., (1994) Structure 2:1121-23) . If two polypeptide chains of a diabody are identical, the resulting diabody when paired contains two identical antigen-binding sites. To obtain a diabody with two different antigen-binding sites, polypeptide chains having different sequences can be used. Likewise, tribodies and tetrabodies are antibodies containing three and four polypeptide chains, respectively, forming three and four antigen-binding sites, respectively, which may be the same or different.

The complementarity determining regions (CDRs) and framework regions (FRs) of a particular antibody can be determined using the system described in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, ^5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242. Although the CDRs disclosed herein are described using Kabat nomenclature, if desired, they may also be redefined according to an alternative nomenclature scheme, such as Chothia (see Chothia & Lesk, (1987) J. Mol. Biol. 196: 901-917; Chothia et al., (1989) Nature 342:878-883 or Honegger & Pluckthun, (2001) J. Mol. Biol. 309:657-670). One or more CDR regions can be incorporated into a molecule by covalent or non-covalent linkage to form an antigen binding protein. An antigen binding protein may include CDR region(s) as part of a larger polypeptide chain, may covalently link said CDR(s) to another polypeptide chain, or may include said CDR(s) through a non-covalent linkage. CDR regions allow an antigen binding protein to specifically bind to a particular antigen of interest.

The antigen binding protein may contain one or more binding sites. If there is more than one binding site, said binding sites may be identical or different. For example, a naturally occurring human immunoglobulin typically contains two identical binding sites, whereas a bispecific or bifunctional antibody contains two different binding sites. Antigen binding proteins of the specified bispecific form include aspects of the present invention.

As used herein, the term human antibody refers to all antibodies containing one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all variable and constant domains are derived from human immunoglobulin sequences (a fully human antibody). These antibodies can be produced by a variety of methods, examples of which are given below, including by immunization with the antigen of interest in a mouse genetically modified to express antibodies derived from genes encoding human heavy and/or light chains; for example, a mouse produced using the Xenomouse®, UltiMab™, HuMAb-Mouse®, Velocimouse®, Velocimmune®, KyMouse or AlivaMab system; or may be derived from a human heavy chain transgene-carrying mouse, a human transgenic rat antibody repertoire, a transgenic rabbit human antibody repertoire, or a human cow antibody repertoire; or they can be produced using HuTarg™ technology. Phage-based techniques may also be used.

A humanized antibody has a sequence that differs from that of an antibody derived from a non-human species by one or more substitutions, deletions and/or additions of amino acid(s) such that the humanized antibody is less likely to induce an immune response and/or induces a less severe immune response response compared to a non-human antibody when administered to a human subject. In one embodiment, certain amino acids in the framework and constant domains of the heavy and/or light chains of a non-human species antibody are altered to produce a humanized antibody. In another embodiment, the constant domain(s) of a human antibody is fused to the variable domain(s) of a non-human species. In another embodiment, one or more amino acid residues of one or more CDR sequences of a non-human antibody

- 8 044919 are modified to reduce the likelihood of said non-human antibody being immunogenic when administered to a human subject, wherein said modified amino acid residues are either not critical for immunospecific binding of said antibody to an antigen, or the amino acid sequence changes made are conservative such that binding of said of a humanized antibody to an antigen is not significantly impaired compared to the binding of a non-human antibody to an antigen. Examples of methods for producing humanized antibodies are given in US patent Nos. 6054297, 5886152 and 5877293.

As used herein, the term chimeric antibody refers to an antibody comprising one or more regions of one antibody and one or more regions of one or more other antibodies. In one embodiment, one or more of the CDR regions is derived from a human antibody that binds to the selected target. In another embodiment, all CDR regions are derived from a human antibody that binds to the selected target. In another embodiment, CDR regions from more than one human antibody that binds to a selected target are mixed and placed into a chimeric antibody. For example, a chimeric antibody may comprise the light chain CDR1 of a first human antibody binding to a selected target, the light chain CDR2 and CDR3 of a second human antibody binding to a selected target, and the heavy chain CDR regions of a third antibody binding to a selected target. Further, the framework regions can be derived from one of the antibodies that bind to the selected target; from one or more other antibodies, for example human antibodies or humanized antibodies. In one example of a chimeric antibody, a portion of the heavy and/or light chain is identical, homologous, or derived from an antibody of a particular species or belonging to a particular class or subclass of antibodies, while the remaining portion of the chain(s) is identical, homologous, or derived from an antibody or antibodies of a different species , or belonging to another class or subclass of antibodies. Also included are fragments of such antibodies that exhibit the desired biological activity (eg, capable of specifically binding to a selected target).

As used herein, the terms Fc and Fc region are used interchangeably and refer to an antibody fragment that contains the C _H2 and C _H3 domains of a human or non-human (e.g., mouse) immunoglobulin, or that contains two contiguous regions that are at least 90% identical domains C _H2 and C _H3 of human or non-human immunoglobulin. These two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of CH3 domains. Fc may, but does not necessarily have to, be capable of interacting with the Fc receptor. See, for example, Hasemann & Capra, Immunoglobulins: Structure and Function, in William E. Paul, ed., Fundamental Immunology, Second Edition, 209, 210-218 (1989), incorporated herein by reference in its entirety.

As used herein, the terms Fc fusion and Fc fusion protein are used interchangeably and refer to a peptide or polypeptide covalently linked to an Fc domain.

As used herein, the terms protein and polypeptide are used interchangeably and mean any chain composed of at least five naturally occurring or non-naturally occurring amino acids linked by a peptide bond.

As used herein, the term peptibody refers to a polypeptide containing one or more bioactive peptides linked, optionally via linkers, to an Fc domain. For examples of peptibodies, see US Patent No. 6660843, US Patent No. 7138370 and US Patent No. 7511012.

As used herein, the term Fab' fragment refers to a structure containing one light chain and part of one heavy chain, which includes the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, so that between the two heavy chains of the two Fab' -fragments, an interchain disulfide bond can be formed to produce an F(ab') ₂ molecule.

As used herein, the term F(ab') ₂ fragment refers to a structure containing two light chains and two heavy chains containing part of the constant region between the _CH 1 and _CH 2 domains, such that an interchain disulfide bond is formed between the two heavy chains . Thus, the F(ab') ₂ fragment consists of two Fab' fragments, which are held together by a disulfide bond between the two heavy chains.

As used herein, the term Fv region refers to a structure containing variable regions of both the heavy and light chains, but not containing constant regions.

As used herein, the term domain antibody refers to an immunologically functional immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region. In some cases, two or more VH regions are covalently linked by a peptide linker to produce a bivalent domain antibody. The targets of these two VH regions of a bivalent domain antibody can be the same antigen or different antigens.

As used herein, the term hemibody refers to an immunologically functional immunoglobulin construct comprising a complete heavy chain, a complete light chain, and a second heavy chain Fc region paired with a full heavy chain Fc region. A linker may, but does not have to be, used to connect the Fc-9044919 heavy chain region and the second heavy chain Fc region. In certain embodiments, the hemibody is a monovalent form of an antigen binding protein described herein. In other embodiments, pairs of charged residues may be used to link one Fc region to a second Fc region.

As used herein, the terms bivalent antigen binding protein or bivalent antibody refer to an antigen binding protein or antibody containing two antigen binding sites. In some cases, the two binding sites have identical antigenic specificity. Bivalent antigen binding proteins and bivalent antibodies can be bispecific, as described herein, and constitute aspects of the present invention.

As used herein, the terms multispecific antigen binding protein or multispecific antibody, when used in the context of a protein component, refer to such a component having more than one target antigen or epitope and forming a further aspect of the present invention.

As used herein, the terms bispecific, dual specificity, or bifunctional, when used in the context of an antigen binding protein or a protein component of an antibody, mean a hybrid antigen binding protein or antibody, respectively, containing two different antigen binding regions. Bispecific antigen binding proteins and antibodies are forms of a multispecific antigen binding protein or a multispecific antibody, and can be produced by various methods, including, but not limited to, hybrid fusion or Fab' fragment joining. See, for example, Songsivilai and Lachmann, (1990) Clin. Exp. Immunol. 79:315-321; Kostelny et al., (1992) J. Immunol. 148:1547–1553. The two binding sites of a bispecific antigen binding protein or antibody bind to two different epitopes, which may be located on the same or different target proteins.

As used herein, the term protein A means any protein identical or substantially similar to protein A of staphylococcus, including commercially available and/or recombinant forms of protein A. For the purposes of the present invention, protein A includes, in particular, media based on recombinant protein A, for example , Mab Select SuRe™ media (GE Healthcare), in which one subunit (e.g., subunit B) is replicated two or more times and linked in a continuous sequence to produce a recombinant Protein A molecule, and other unnatural Protein A molecules.

As used herein, the term G protein means any protein identical or substantially similar to the streptococcal G protein, including commercially available and/or recombinant forms of the G protein. Proteins A and G are often used to purify antigen-binding proteins (e.g., antibodies, peptibodies, and other hybrid proteins containing an Fc region) using affinity chromatography. See, for example, Vola et al. (1994), Cell Biophys. 24-25: 27-36; Aybay and Imir (2000), J. Immunol. Methods 233(1-2): 77-81; Ford et al. (2001), J. Chromatogr. In 754: 427-435. Proteins A and G are suitable for this purpose because they bind to the Fc region of these types of proteins. Recombinant fusion proteins containing the Fc region of an IgG antibody can be purified using similar methods. Proteins A and G can be used in the described methods as an adsorbent component of the separation matrix.

As used herein, the terms recover and purify are used interchangeably and mean a reduction of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% and 99%, or more, of the amount of heterogeneous elements, for example, biological macromolecules such as proteins or DNA, that may be present in a sample containing the protein of interest. The presence of heterogeneous proteins can be determined using any suitable analytical method, including high performance liquid chromatography (HPLC), gel electrophoresis and staining and/or ELISA. Analysis for the presence of DNA and other nucleic acids can be performed by any suitable method, including the use of gel electrophoresis and staining, and/or the use of polymerase chain reaction.

As used herein, the terms protectant and additive are used interchangeably and mean a compound capable of limiting or modulating the degree of modification of proteins in response to a dose of UVC. A non-limiting list of protectants suitable for use in the described methods includes any one or more of tyrosine, tryptophan, methionine, pyridoxine and riboflavin. The term protectant covers individual compounds as well as combinations of compounds such as tyrosine and tryptophan, which may be present in any ratio relative to each other. According to the description given in this application, tyrosine makes the smallest contribution to the total UV absorption.

As used herein, the term UV dose refers to the amount of energy delivered to the target in the form of UV radiation. The dose of UV radiation delivered to the target is

- 10 044919 is a function of the intensity and time of exposure. Non-limiting examples of UV dose include about 1 mJ/ ^cm2 , about 10 mJ/ ^cm2 , about 25 mJ/ ^cm2 , about 50 mJ/ ^cm2 , about 75 mJ/ ^cm2 , about 100 mJ/ ^cm2 , approximately 125 mJ/ ^cm2 , approximately 200 mJ/ ^cm2 , approximately 250 mJ/ ^cm2 , approximately 300 mJ/cm2, approximately 350 mJ/ ^cm2 , approximately 400 mJ/ ^cm2 , approximately 450 mJ/ ^{cm2 ,} approximately 500 mJ/cm ² , about 600 mJ/cm ² , about 700 mJ/cm ² , about 800 mJ/cm ² , about 900 mJ/cm ² , about 1000 mJ/cm ² and more than about 1000 mJ/cm ² .

As used herein, the term UV radiation refers to a region of the light spectrum including wavelengths in the range of at least 10 nm and up to a maximum of 400 nm. For example, the term UV radiation covers light having a wavelength in the range from about 200 nm to about 280 nm, including a wavelength of about 254 nm. According to the methods proposed in this application, UV radiation can be supplied in the form of uniform collimated and filtered light; Accordingly, the term UV radiation covers both uniform collimated and uncollimated UV radiation, as well as filtered UV radiation and unfiltered UV radiation.

As used herein, the term protein component-containing sample means an aliquot of liquid containing at least an aqueous component and a protein component. According to various embodiments, said aqueous component may comprise a buffer. Said protein component may comprise any substance containing two or more amino acids linked by a peptide bond. The protein component may contain some or all of the 20 naturally occurring amino acids, and the remainder of the protein component may contain any non-naturally occurring amino acid. Accordingly, the methods proposed herein can be implemented in relation to a sample containing a protein containing one or more naturally occurring amino acids, or one or more non-naturally occurring amino acids. In various embodiments, the protein in the sample containing the protein component is an antigen binding protein comprising any one or more of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a single chain antibody, a diabody, a tribody, a tetrabody, Fab fragment, F(ab') ₂ fragment, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody or IgG4 antibody and fragments thereof, Fc domain; peptide; a hybrid protein with an Fc fragment and a therapeutic protein.

As used herein, the term reactive substances means any component of a solution that is capable or capable of reacting with another component, resulting in modification or oxidation of the reacted component. Non-limiting examples of reactive species include oxygen ions (eg, ^O2- ), hydroxyl ions (eg, OH-), and _peroxides (eg, _H2O2 ).

As used herein, the term water number is a value calculated using the following equation:

l-lQ-αί

Water number = ------, t in (ίο) where a = absorption capacity of the solution and l is the path length in meters.

II. Method for inactivating the virus in a sample.

Viral inactivation is a critical step in the preparation of protein solutions for therapeutic use. Moreover, viral inactivation standards have been established by various regulatory authorities, and many manufacturers have attempted to address this issue. Viral inactivation technologies have evolved along several lines, including filtration technology, high-temperature short-term treatment (HTST) technology, and UVC. Although each of these technologies has its own strengths, each of them also has disadvantages. In the case of UVC, it has been noted that long-term exposure of proteins to UVC radiation, while an effective and efficient way to inactivate viruses, may nevertheless lead to protein degradation and/or oxidation. Accordingly, although UVC technology is an effective method for inactivating viruses, exposing a protein-containing sample to high doses of UVC radiation can lead to undesirable effects on the protein itself. Accordingly, according to one aspect of the described invention, a method is provided that allows the use of high doses of UVC necessary to inactivate the virus in a sample containing a protein component, while reducing or completely eliminating the possibility of damage to the protein itself. Accordingly, a method has been proposed for inactivating a virus in a sample containing a protein component. According to one embodiment, this method can be implemented as follows.

First, a sample containing the protein component is obtained; it is known or assumed that the specified sample contains a virus. The specified sample containing the protein component can have any composition, with the proviso that the specified sample contains protein. For example, specified

- 11 044919 This sample may contain eluent from the chromatographic column collected in a pool. According to this embodiment, the pool obtained on the chromatography column can be collected during any chromatographic manipulation. Examples of chromatography column-derived pools include a Protein A column eluent pool containing a protein component, a Protein G column eluent pool containing a protein component obtained with a hydrophobic interaction chromatography (GC) column, a hydrophobic interaction chromatography (GC) column-containing pool containing a protein component a size exclusion chromatography (SEC) column pool containing the protein component, an ion exchange chromatography (IEC) column pool containing the protein component, and a hydroxyapatite column pool containing the protein component.

A sample containing a protein component may also contain an eluent exiting the chromatography column. For example, said output eluent may be obtained from a chromatography column; Accordingly, this method can be implemented in situ and in real time. Examples of chromatographic eluents include a protein component A column effluent containing a protein component, a protein G column effluent containing a protein component effluent from a hydrophobic interaction chromatography (GC) column, a protein component effluent from a size exclusion chromatography column ( SEC) a protein component-containing effluent, an ion exchange chromatography (IEC) column effluent containing a protein component, and a hydroxyapatite column effluent containing a protein component.

Although the described method can be applied to a sample containing any type of protein component, the described method may be particularly advantageous in the context of a protein therapeutic, which is an area where viral inactivation standards have been adopted. Accordingly, in one example, the sample containing the protein component is a sample containing a protein-based pharmaceutical molecule. In specific embodiments, the protein component of a sample according to the described method comprises an antigen binding protein (e.g., any one or more of (i) an antigen binding protein comprising any one or more of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, single chain antibody, diabody, tribody, tetrabody, Fab fragment, F(ab') ₂ fragment, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody or IgG4 antibody, and their fragments, (ii) Fc domain; (iii) peptide; (iv) fusion protein with Fc fragment and (v) therapeutic protein), Fc domain, peptide and therapeutic protein. These types of molecules are common attributes of therapeutic molecules. With respect to antigen binding protein antibodies, as used herein, the term antibody includes fully human antibodies, humanized antibodies, or fully non-human antibodies (eg, mouse antibodies), and the described method can be applied to all of these types of molecules.

According to various embodiments of the described method, the sample being processed using the described methods may be a sample containing cells in which the virus is desired to be inactivated. Examples of such samples include a sample containing platelets, CHO cells, or bacterial cells such as E. coli in which viruses are desired to be inactivated. Such a sample may contain cell cultures. According to these embodiments, the specified method can be implemented in accordance with the above description, replacing the sample containing cells with a sample containing a protein component.

UVC viral inactivation is most often applied to samples containing a protein component or a sample containing cells such as platelets, although this is not a requirement; in other embodiments, the described methods can also be used to remove virus from a sample that does not contain a protein component.

In one aspect, the described methods are aimed at inactivating viruses that may be inadvertently introduced into samples containing a protein component. Possible sources of unintentional introduction of viruses into the protein production process include contaminated raw materials or contact with production personnel. One of the advantages of the described methods is that they are suitable for viruses of any type, and regardless of whether the specified virus has an envelope or does not have an envelope. Thus, this method can be applied to double-stranded DNA viruses, single-stranded DNA viruses, double-stranded RNA viruses and single-stranded RNA viruses. Examples of families of viruses, by default including all members of a specified family, that can be inactivated using the methods described include adenoviridae, asfarviridae, herpesviridae, iridoviridae, papillomaviridae, polyomaviridae, poxviridae, circoviridae, hepadnaviridae, parvoviridae, birnaviridae, reoviridae, arenaviridae, vornaviridae, bunyaviridae, deltaviridae, filoviridae, orthomyxoviridae, paramyxoviridae, rhabdoviridae, arterioviridae, astroviridae, caliciviridae, coronaviridae, flaviviridae, hepatitis E-like viruses, nodaviridae, picornaviridae, togaviridae and tertroviridae. In specific embodiments that may be particularly suitable for use in methods for producing therapeutic proteins, viruses that can be inactivated using the described methods include MVM parvovirus, MuLV retrovirus, or CVV bunyavirus.

Following this method further, the target dose of UV radiation is determined, under the influence of which the virus is inactivated. In order to most effectively and efficiently inactivate the virus using UVC, it is desirable to determine the target dose of UVC that will provide the required result. Although the described methods can be implemented without optimizing the UVC exposure conditions (which collectively constitute the UVC dose) for the type of virus to be inactivated, and any convenient dose of UVC can be used to implement the specified method, the effectiveness of the specified method can be increased by determining the target dose specific for the virus to be inactivated. It is noted that for some viruses the conditions for inactivation by UV radiation are the same, and it is possible to inactivate two or more types of viruses using a single procedure according to the described method, selecting suitable exposure conditions. Various studies have been conducted to determine the UV sensitivity of various DNA and RNA viruses. See, for example, Lytle & Sagripanti, (2005) J Virol. 79:14244-252, and Knipe et al., (2007) Field's Virology, Lippincott Williams & Wilkins, incorporated herein by reference, and FIG. 11 and 12.

A preservative is then added to the sample to obtain a stabilized mixture. One of the functions of the protectant is to trap reactive substances that can degrade or modify sample components, such as protein in the sample, to reduce or completely prevent any modifications or degradation that may occur as a result of exposing the sample to UVC radiation. In particular, exposing a solution to doses of UVC radiation required for a specific procedure, for example, to inactivate viruses, can lead to the formation of reactive substances. In some cases, the presence of these reactive substances in the sample may lead to undesirable modifications, for example, due to indirect oxidation of sample components, including proteins. In other cases, the presence of reactive substances in the sample may also contribute to indirect modification of sample components. As noted herein, the potential for modification and/or degradation of proteins is one of the concerns associated with the use of UVC radiation in viral inactivation methods.

Examples of reactive species capable of degrading or modifying proteins include reactive species such as oxygen ions (eg, O ^{2 -} ), hydroxyl ions (eg, OH ^- ) and peroxides (eg, H ₂ O ₂ ). Because these and other reactive substances are often formed when a solution is exposed to high doses of UV radiation, which are often necessary for effective viral inactivation, it is preferable to add a protectant before exposing the sample to UV radiation.

Not all chemicals can be used as a protective agent. In fact, detailed research was carried out to determine suitable protective agents as outlined in the Examples provided. A suitable preservative is a compound that has the ability to capture any reactive substances present in the sample, such as those produced by exposure to UV light, such that the effect of said reactive substances on components of the sample (eg, protein) is reduced relative to the effect of said reactive substances on the components sample in the absence of a protective agent. In some cases, degradation or modification of sample components due to exposure to reactive substances generated during the UVC procedure can be prevented by the use of a preservative.

In addition to its effectiveness in neutralizing the undesirable effects of any reactive substances present in a sample, the choice of a protective agent must also take into account the difficulty associated with its removal from the sample after exposure to UVC. When implementing the described method in relation to a sample containing a therapeutic molecule, such as an antigen binding protein (for example, any one or more of (i) an antigen binding protein containing any one or more of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, recombinant antibody, single chain antibody, diabody, tribody, tetrabody, Fab fragment, F(ab') ₂ fragment, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody or IgG4 -antibodies, and fragments thereof, (ii) an Fc domain; (iii) a peptide; (iv) a fusion protein with an Fc fragment; and (v) a therapeutic protein) or therapeutic protein, this consideration becomes of great importance. Due to regulatory restrictions imposed on product quality, a desirable property of a protective agent is the ability to remove it from the sample after the protective function has been implemented.

A list of suitable protective agents is proposed, taking into account all the properties necessary for a protective agent described above; it includes, but is not limited to, tyrosine, tryptophan, methionine, pyridoxine and riboflavin. In various embodiments, the protectant contains two or more compounds in varying proportions. For example, a protective agent

- 13 044919 may contain tyrosine, tryptophan, or both tyrosine and tryptophan in any desired proportions. The exact composition and proportions in the combination of protective agents can be determined empirically and/or as described herein.

Additional options for protective devices can be easily determined using the present description as a guide. In one such screening option, a potential protective agent may be added to a protein-containing sample exposed to UV light at a dose suitable to inactivate one or more viruses (FIGS. 11 and 12, and references cited herein may be used as a guide in determining the appropriate dose of UVC); an analysis is then performed to determine the degree of degradation or modification of the protein. For this purpose, standard chromatographic and analytical techniques can be used. For example, IOC can be used to assess protein modification, and SEC or mass spectroscopy can be used to analyze protein degradation.

The protective agent used in the described methods can be added in any concentration. In a preferred embodiment, the preservative is added to a concentration that effectively reduces or eliminates modification or degradation of the sample component. The amount of protective agent can be determined in a similar manner to identifying the protective agent, that is, the selected protective agent can be added to the sample at an initial concentration, said sample is exposed to UV light, and the degree of degradation and/or modification of the sample component (e.g., protein) is determined with using the generally accepted method. As with protective agent identification, suitable techniques include IOC, SEC and mass spectroscopy. If the required degree of protection for the protein component is not achieved, the evaluation may be repeated until the concentration that provides the required degree of protection is determined. In a specific embodiment, the protectant is added to the sample at a concentration ratio of greater than 1 part protectant to 200 parts protein. In other words, the specified protective agent can be added to the sample at a relative concentration of more than 1 mm protective agent per 200 mm protein (ie, 1:200). Other suitable relative concentrations include 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:90, 1:80 , 1:70, 1:60, 1:50, 1:40, 1:30, 1:20 or 1:10.

Although the proposed protectants and protectant concentrations as described are suitable for use, additional protectant options and protectant concentrations can be easily determined using an empirical matrix method. In one example of this approach, a matrix may be constructed with one axis representing the various potential protective agents and the other axis representing the various concentration levels. To populate the matrix with preferred protectants and preferred concentrations of said protectants, experiments may be performed as described herein (eg, using SEC, IOC, and/or mass spectroscopy to evaluate the effect of a particular protectant and a particular concentration). This approach will allow us to discover additional variants of protective agents and concentrations of protective agents.

After obtaining a stabilized mixture containing a sample containing a protein component and a protective agent, the stabilized mixture is exposed to UV radiation from a source operating at a selected power level and wavelength for a selected period of time. To denote the combination of these parameters in this application, the collective term UVC dose is used. Examples of sources suitable for use in the described method include Newport Oriel® Flood UVC sources, such as model 97536.

The UVC source is preferably configured to operate over a range of power levels. Preferred power levels range from about 1 mJ to about 1000 mJ. In specific examples, the UVC source is capable of providing approximately 1 mJ, approximately 10 mJ, approximately 25 mJ, approximately 50 mJ, approximately 75 mJ, approximately 100 mJ, approximately 125 mJ, approximately 200 mJ, approximately 250 mJ, approximately 300 mJ, approximately 350 mJ , approximately 400 mJ, approximately 500 mJ, approximately 600 mJ, approximately 700 mJ, approximately 800 mJ, approximately 900 mJ or approximately 1000 mJ. It is also desirable to be able to switch the UVC source from the first power level to the second power level, either automatically in response to feedback from the sensor, or manually by the operator.

The UVC source is also preferably configured to deliver UVC radiation in a specific wavelength range. The preferred wavelength range is from about 200 nm to about 280 nm, which corresponds to the full range of the C region of the UV spectrum. In specific preferred embodiments, the wavelength is approximately 254 nm.

When a protectant or combination of protectants is added to a sample to form a stabilized mixture, the absorbency of said stabilized mixture may change compared to the absorbency of the sample without the addition of the protectant. For example, added protective agent(s) or other compounds present in the stabilized sample (eg, buffer components, solubilizing agents, etc.) may absorb some of the UV radiation incident on the sample. This may result in a reduction in the effective amount of UV radiation received by any virus present in the sample and, as a result, a reduction in the effectiveness of viral inactivation.

Feedback may be used to account for the inherent absorptive capacity of the protectant(s) and/or other components of the solution and to ensure that the stabilized mixture has received the target dose of UV radiation, allowing the UV exposure parameters to vary based on the assessment absorption of UV radiation by the mixture. Accordingly, after assessing the absorptivity of the mixture entering the UVC irradiation device, radiation parameters within the device (e.g., lamp power, residence time, etc.) can be temporarily modified to ensure that the target dose is delivered to the stabilized mixture exiting the device. Such an assessment may alternatively be carried out by measuring the absorption capacity of the mixture leaving the device, or by measuring the parameters of the mixture inside the device. In one embodiment, such an assessment can be performed by monitoring the absorbance of the sample at a given wavelength, for example, 254 nm. Absorbance readings can be used to determine the dose received, and can be defined as adjusted energy dose delivery data that takes into account the absorbance of components that may be present in the sample (for example, protein, protective chemicals, or other chemicals present in the solution) with respect to ultraviolet radiation. According to one embodiment, this assessment can be performed by measuring the power of the radiation source on the surface of the solution. Alternatively, the power of the radiation source can be measured at the surface of the lamp. Also, to estimate the power of the radiation source, the electrical power consumed by the lamp can be measured.

This assessment is expected to determine the reduction in received dose due to the absorbency of the protective agent(s) in the stabilized sample. Accordingly, any one or more of wavelength, UV source power, and UV exposure time are then modulated if the stabilized mixture is judged to have not received a target dose of UV radiation.

In one embodiment, for a highly agitated vessel receiving a collimated beam of UVC radiation, the dose can be adjusted by applying the formula:

Water number=------, al In (10)’ where a = absorptive capacity of the solution and l is the path length. See, for example, Bolton, (2003) ASCE, 129(3): 209-215. Once the water number for a particular viral inactivation procedure is determined, the target UV dose is divided by the specified water number to determine the exposure time for the treatment. In another embodiment, for an unstirred vessel into which UVC radiation is applied (e.g., a thin film reactor or the like), the dose can be adjusted by applying the formula:

dose ~ (P / Q)exp(-al) = (Po / Qo)exp(-aol), where a = absorption capacity of the solution, and l is the path length along the annular gaps. In the above formula, P represents the output power of the lamp, and Q represents the volumetric flow rate of the mixture with absorption capacity = a; P ₀ and Q ₀ represent the power and flow rate of the reference mixture with absorption capacity = a ₀ . See, for example, Ye, Z (2007) UV Disinfection Between Concentric Cylinders (PhD thesis, Georgia Institute of Technology).

It should be noted that any or all steps of the described methods can be performed manually or in any convenient automated manner, for example, using automated or computerized systems. In some embodiments, the process may be fully automated. In other embodiments, one or more steps may be automated. For example, measuring the delivered dose and modulating in response to changes in target dose levels may be one automated step. In one embodiment, the stabilized sample is exposed to UV light while deviations from the target dose are monitored. If deviations from the target dose are detected, the control module is able to adjust the exposure time, radiation wavelength, or UV source power to ensure the target dose is delivered. This can happen in real time using a feedback element.

The described method can be implemented at any scale, and either as a separate procedure or as a continuous flow process. According to one embodiment of a separate procedure, a stabilized sample of any volume is formed in the vessel. Said vessel is then exposed to UV radiation (e.g. UVC radiation) and then assessed for inactivation

- 15 044919 viruses. This procedure can be repeated until any viruses present in the sample are inactivated. Alternatively, this assessment can be carried out continuously simultaneously with exposure to UV radiation. Once the virus is inactivated, the sample can be transferred to another vessel for further processing or packaging.

In an embodiment of a continuous flow process, a stabilized sample may be obtained from the effluent from a previous purification step, with a protective agent added to the effluent as it flows from the first column. The preservative may be mixed with the effluent stream due to any shear forces associated with the introduction of the preservative into the effluent stream. The stabilized sample can then be passed through a device configured to continuously expose the sample passing through it to UV light. After delivery of the target dose of UV radiation, the effluent can be sent to a second purification step, for example, to remove undesired protective agent(s) or other compounds present in the stabilized sample.

According to another aspect, the described method can be implemented on any scale, from laboratory scale to industrial scale. When implementing this method on an industrial scale, it may be advisable to divide the stabilized sample into aliquots and parallel processing of each aliquot. For example, multiple UVC sources can operate in parallel to provide processing of large volumes of stabilized sample. In fig. 13 shows a schematic example of such a configuration.

III. A method of reducing protein degradation or modification caused by the presence of reactive substances formed when exposed to UV.

According to the description given in this application and FIG. 1-8, a sample containing a protein component may be degraded or modified when exposed to UV radiation, in particular radiation in the UV range. The UVC region, however, is the region of the spectrum that is most effective for various purposes, such as virus inactivation, and is particularly suitable for industrial applications. The observed degradation and/or modification of the protein may be due to the presence of reactive substances that are formed when the dissolving component of the sample is irradiated with ultraviolet or ionizing radiation. Examples of reactive species include oxygen ions (eg, O ^{2 -} ), hydroxyl ions (eg, OH ^- ) and peroxide compounds (eg, H ₂ O ₂ ). The presence of reactive substances can have a destructive effect on proteins. See, for example, Cabiscol, et al., (2010) Int. Microbiol 3:315 and Bandyopadhyay et al. (1999) Curr. Sci. 77:658-666.

During the protein production procedure, UV radiation can be used to inactivate viruses, but it can also promote degradation and/or modification of the protein. Accordingly, in one aspect, the invention described herein provides a method for reducing protein degradation or modification caused by the presence of reactive substances generated by exposure to UV. According to one embodiment, the described method can be implemented as follows.

First, a sample is obtained containing a protein component that is known or suspected to decompose or undergo modification in the presence of reactive substances. The sample containing the protein component can have any composition, with the proviso that the specified sample contains protein. For example, said sample may contain a pool of chromatography column eluent. According to this embodiment, the pool obtained on the chromatography column can be collected during any chromatographic manipulation. Examples of chromatography column-derived pools include a Protein A column eluent pool containing a protein component obtained from a Protein G column eluent pool containing a protein component obtained from a hydrophobic interaction chromatography (GC) column a pool containing a protein component obtained from a GC column a size exclusion chromatography (SEC) pool containing a protein component, an ion exchange chromatography (IEC) column pool containing a protein component, and a hydroxyapatite column pool containing a protein component.

A sample containing a protein component may also contain an eluent exiting the chromatography column. For example, said output eluent may be obtained from a chromatography column; Accordingly, this method can be implemented in situ and in real time. Examples of chromatographic eluents include a protein component A column effluent containing a protein component, a protein G column effluent containing a protein component effluent from a hydrophobic interaction chromatography (GC) column, a protein component effluent from a size exclusion chromatography column ( An EC stream containing a protein component, an ion exchange chromatography (IEC) column effluent containing a protein component, and a hydroxyapatite column effluent containing a protein component.

Although the described method can be applied to a sample containing any type of protein component, the described method may be particularly advantageous in the context of protein-based therapeutics, an area where protein degradation and/or modification may be significant. difficulties. Accordingly, in one example, the sample containing the protein component is a sample containing a protein-based pharmaceutical molecule. In specific embodiments, the protein component of a sample according to the described method comprises an antigen binding protein (e.g., any one or more of (i) an antigen binding protein comprising any one or more of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, single chain antibody, diabody, tribody, tetrabody, Fab fragment, F(ab') ₂ fragment, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, 1gG2 antibody, IgG3 antibody or IgG4 antibody antibodies, and fragments thereof, (ii) Fc domain; (iii) peptide; (iv) fusion protein with Fc fragment and (v) therapeutic protein), Fc domain, peptide and therapeutic protein. These types of molecules are common attributes of therapeutic molecules. With respect to antibodies, as used herein, the term antibody includes fully human antibodies, humanized antibodies, or fully non-human antibodies (eg, mouse antibodies); The described method can be applied to all of these types of molecules.

According to various embodiments of the described method, the sample being processed using the described methods may be a sample containing cells in which the virus is desired to be inactivated. Examples of such samples include a sample containing platelets, CHO cells, or bacterial cells such as E. coli in which viruses are desired to be inactivated. Such a sample may contain cell cultures. According to these embodiments, the specified method can be implemented in accordance with the above description, replacing the sample containing cells with a sample containing a protein component.

Following this method further, the target dose of UV radiation is determined. The target dose may be selected on any basis, however, in one preferred embodiment, the target dose is the dose at which the virus of interest is inactivated. When choosing a target dose that corresponds to the UV dose for known or probable inactivation of, for example, a specific virus of interest, for the most effective and efficient inactivation of the virus using UVC, it is desirable to determine the target dose of UVC that will provide the required result. Although the described methods can be implemented without optimizing the UVC exposure conditions (which collectively constitute the UVC dose) for the type of virus to be inactivated, and any convenient dose of UVC can be used to implement the specified method, the effectiveness of the specified method can be increased by determining the target dose specific for the virus to be inactivated. It is noted that for some viruses the conditions for inactivation by UV radiation are the same, and by selecting suitable exposure conditions, it is possible to inactivate two or more types of viruses using a single procedure according to the described method. Various studies have been conducted to determine the UV sensitivity of various DNA and RNA viruses. See, for example, Lytle & Sagripanti, (2005) J Virol. 79:14244-252, and Knipe et al., (2007) Field's Virology, Lippincott Williams & Wilkins, incorporated herein by reference, and FIG. 11 and 12.

Then, following the following method, a preservative is added to the sample to obtain a stabilized mixture. One of the functions of the protectant is to trap reactive substances that can degrade or modify sample components, such as protein in the sample, to reduce or completely prevent any modifications or degradation that may occur as a result of exposing the sample to UVC radiation. In particular, exposure of the solution to the required doses of UV radiation can lead to the formation of reactive substances as described in this application. Indeed, this is one of the problems associated with the use of UVC radiation in viral inactivation methods. The presence of these reactive substances in the sample can lead to indirect oxidation of sample components, including proteins. In addition, the presence of reactive substances may also contribute to indirect modification of sample components.

Examples of reactive species capable of degrading or modifying proteins include reactive species such as oxygen ions (eg, O ^{2 -} ), hydroxyl ions (eg, OH ^- ) and peroxides (eg, H ₂ O ₂ ). Because these and other reactive substances are often formed when a solution is exposed to high doses of UV radiation, which are often necessary for effective viral inactivation, it is preferable to add a protectant before exposing the sample to UV radiation.

Not all chemicals can be used as a protective agent. Moreover, detailed research was carried out to determine suitable protective agents as outlined in the examples given.

A suitable preservative is a compound that has the ability to capture any reactive substances present in the sample, such as those formed by exposure to UV light, so that the effect of said reactive substances on the sample components

- 17 044919 (for example, on protein) is reduced compared to the effect of said reactive substances on the sample components in the absence of a protective agent. In some cases, degradation or modification of sample components due to exposure to reactive substances generated during the UVC procedure can be completely prevented by the use of a preservative.

In addition to its effectiveness in neutralizing the undesirable effects of any reactive substances present in a sample, the choice of a protective agent must also take into account the difficulty associated with its removal from the sample after exposure to UVC. When implementing the described method in relation to a sample containing a therapeutic molecule, such as an antigen binding protein (for example, any one or more of (i) an antigen binding protein containing one or more of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, single chain antibody, diabody, tribody, tetrabody, Fab fragment, F(ab') ₂ fragment, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, 1gG2 antibody, IgG3 antibody or IgG4 antibody, and fragments thereof, (ii) an Fc domain; (iii) a peptide; (iv) a fusion protein with an Fc fragment; and (v) a therapeutic protein) or a therapeutic protein, this issue becomes of great importance. Due to regulatory restrictions imposed on product quality, a desirable property of a protective agent is the ability to remove it from the sample after the protective function has been implemented.

A list of suitable protective agents is proposed, taking into account all the required properties of the protective agent described above; it includes, but is not limited to, tyrosine, tryptophan, methionine, pyridoxine and riboflavin. In various embodiments, the protectant contains two or more compounds in varying proportions. For example, the protectant may contain tyrosine, tryptophan, or both tyrosine and tryptophan in any desired proportions. The exact composition and proportion of the combination of protective agents can be determined empirically and/or as described herein.

Additional protective options can be readily identified using the present disclosure as a guide. In one such screening option, a potential protective agent may be added to a protein-containing sample exposed to UV light at a dose suitable to inactivate one or more viruses (FIGS. 11 and 12, and references cited herein may be used as a guide in determining the appropriate dose of UVC); an analysis is then performed to determine the degree of degradation or modification of the protein. For this purpose, standard chromatographic and analytical techniques can be used. For example, IOC can be used to assess protein modification, and SEC or mass spectroscopy can be used to analyze protein degradation.

The protective agent used in the described methods can be added in any concentration. In a preferred embodiment, the preservative is added to a concentration that effectively reduces or eliminates modification or degradation of the sample component. The amount of protective agent can be determined in a manner similar to identifying the protective agent, that is, the selected protective agent can be added to the sample at an initial concentration, said sample is exposed to UV light, and the degree of degradation and/or modification of the sample component (e.g., protein) is determined with using the generally accepted method. As with protective agent identification, suitable techniques include IOC, SEC and mass spectroscopy. If the required degree of protection for the protein component is not achieved, the evaluation may be repeated until the concentration that provides the required degree of protection is determined. In a specific embodiment, the protectant is added to the sample at a concentration ratio of greater than 1 part protectant to 200 parts protein. In other words, the specified protective agent can be added to the sample at a relative concentration of more than 1 mm protective agent per 200 mm protein (ie 1:200). Other suitable relative concentrations include 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:90, 1:80 , 1:70, 1:60, 1:50, 1:40, 1:30, 1:20 or 1:10.

Although the proposed protectants and protectant concentrations as described herein are suitable for use, additional protectant options and protectant concentrations can be easily determined using the empirical matrix method. In one example of this approach, a matrix may be constructed with one axis representing the various potential protective agents and the other axis representing the various concentration levels. To populate the matrix with preferred protectants and preferred concentrations of said protectants, experiments can be performed as described herein (eg, using SEC, IOC, and/or mass spectroscopy to evaluate the effect of a particular protectant and a particular concentration). This approach will allow us to discover additional variants of protective agents and concentrations of protective agents.

After obtaining a stabilized mixture containing a sample containing a protein component and a protective agent, said stabilized mixture is exposed to UV radiation,

- 18 044919 coming from a source operating at a selected power level and at a selected wavelength for a selected period of time. To denote the combination of these parameters in this application, the collective term UVC dose is used. Examples of sources suitable for use in the described method include Newport Oriel® Flood UVC sources, such as model

97536.

The UVC source is preferably configured to operate over a range of power levels. Preferred power levels range from about 1 mJ to about 1000 mJ. In specific examples, the UVC source is capable of delivering approximately 1 mJ, approximately 10 mJ, approximately 25 mJ, approximately 50 mJ, approximately 75 mJ, approximately 100 mJ, approximately 125 mJ, approximately 200 mJ, approximately 250 mJ, approximately 300 mJ, approximately 350 mJ, approximately 400 mJ, approximately 450 mJ, approximately 500 mJ, approximately 600 mJ, approximately 700 mJ, approximately 800 mJ, approximately 900 mJ, approximately 1000 mJ or more than 1000 mJ. It is also desirable to be able to switch the UVC source from the first power level to the second power level, either automatically in response to feedback from the sensor, or manually by the operator.

The UVC source is also preferably configured to deliver UVC radiation in a specific wavelength range. The preferred wavelength range is from about 200 nm to about 280 nm, which corresponds to the full range of the C region of the UV spectrum. In specific preferred embodiments, the wavelength is approximately 254 nm.

When a protectant or combination of protectants is added to a sample to form a stabilized mixture, the absorbency of said stabilized mixture may change compared to the absorbency of the sample without the addition of the protectant. For example, added protective agent(s) or other compounds present in the stabilized sample (eg, buffer components, solubilizing agents, etc.) may absorb some of the UV radiation exposed to the sample. This may result in a reduction in the effective amount of UV radiation received by any virus present in the sample and, as a result, a reduction in the effectiveness of viral inactivation.

Feedback may be used to account for the inherent absorptive capacity of the protectant(s) and/or other components of the solution and ensure that the target dose of UVB radiation has been delivered to the stabilized mixture to allow the UV exposure parameters to vary depending on from assessing the absorption of UV radiation by the mixture. Accordingly, after assessing the absorptive capacity of the mixture entering the UVC irradiation device, radiation parameters within the device (e.g., lamp power, residence time, etc.) can be temporarily modified to ensure delivery of the target dose to the stabilized mixture exiting the device. Such an assessment may alternatively be carried out by measuring the absorption capacity of the mixture leaving the device, or by measuring the parameters of the mixture inside the device. In one embodiment, such an assessment can be performed by monitoring the absorbance of the sample at a given wavelength, for example, 254 nm. Absorbance values can be used to determine the dose received, and can be defined as adjusted energy dose data that takes into account the ability of components that may be contained in the sample (for example, protein, protective chemicals, or other chemicals present in the solution) to absorb ultraviolet light. radiation. According to one embodiment, this assessment can be performed by measuring the power of the radiation source on the surface of the solution. Alternatively, the power of the radiation source can be measured at the surface of the lamp. Also, to estimate the power of the radiation source, the electrical power consumed by the lamp can be measured.

It is expected that this assessment will evaluate the reduction in received dose due to the absorption capacity of the protective agent(s) in the stabilized sample. Accordingly, any one or more of wavelength, UV source power, and UV exposure time are then modulated if the stabilized mixture is assessed to have not received a target dose of UV irradiation.

In one embodiment, for a highly agitated vessel receiving a collimated beam of UVC radiation, the dose can be adjusted by applying the formula:

Water number=------, al In (10)’ where a = absorptive capacity of the solution and l is the path length. See, for example, Bolton, (2003) ASCE, 129(3): 209-215. Once the water number for a particular viral inactivation procedure is determined, the target UV dose is divided by the specified water number to determine the exposure time for the treatment. In another embodiment, for an unstirred vessel receiving UVC radiation (e.g., a thin film reactor or the like), the dose may be adjusted to

- 19 044919 using the formula:

dose ~ (P / Q)exp(-al) = (Po / Qo)exp(-aol), where a = absorption capacity of the solution, and l is the path length along the annular gaps. In the above formula, P represents the output power of the lamp; Q represents the volumetric flow rate of the mixture with absorption capacity = a; P ₀ and Q0 represent the power and flow rate of the reference mixture with absorption capacity = a ₀ . See, for example, Ye, Z (2007) UV Disinfection Between Concentric Cylinders (PhD thesis, Georgia Institute of Technology).

It should be noted that any or all steps of the described methods can be performed manually or in any convenient automated manner, for example, using automated or computerized systems. In some embodiments, the process may be fully automated. In other embodiments, one or more steps may be automated. For example, measuring the delivered dose and modulating in response to changes in target dose levels may be one automated step. In one embodiment, the stabilized sample is exposed to UV light while deviations from the target dose are monitored. If deviations from the target dose are detected, the control module is able to modulate exposure time, radiation wavelength, or UV source power to ensure the target dose is delivered. This can happen in real time using a feedback element.

The described method can be implemented at any scale, either as a separate procedure or as a continuous flow process. According to one embodiment of a separate procedure, a stabilized sample of any volume is formed in the vessel. The vessel is then exposed to UV radiation (eg, UVC radiation) and then assessed for virus inactivation. This procedure can be repeated until any viruses present in the sample are inactivated. Alternatively, this assessment can be carried out continuously simultaneously with exposure to UV radiation. Once the virus is inactivated, the sample can be transferred to another vessel for further processing or packaging.

In a continuous flow process embodiment, the stabilized sample may be obtained from the effluent obtained from the previous purification step, wherein the protective agent is added to the effluent as it exits the first column. The preservative may be mixed with the effluent due to any shear forces associated with the introduction of the preservative into the effluent. The stabilized sample can then be passed through a device configured to continuously expose the sample passing through it to UV light. After delivery of the target dose of UV radiation, the effluent can be sent to a second purification step, for example, a purification step to remove undesired protective agent(s) or other compounds present in the stabilized sample.

According to another aspect, the described method can be implemented on any scale, from laboratory to industrial scale. When implementing this method on an industrial scale, it may be advisable to divide the stabilized sample into aliquots and parallel processing of each aliquot. For example, multiple UVC sources can operate in parallel to provide processing of large volumes of stabilized sample. In fig. 13 shows a schematic example of such a configuration.

IV. A method of reducing the oxidation of methionine residues, tryptophan residues, or both methionine residues and tryptophan residues in a protein exposed to UV radiation.

According to another aspect of the present invention, a method is provided for reducing the oxidation of methionine residues, tryptophan residues, or both methionine residues and tryptophan residues in a protein exposed to UV radiation. As described herein and relevant published data, methionine and tryptophan residues are susceptible to oxidation and may cause protein inactivation. See, for example, Schoneich, (2005) Biochim Biophys Acta 1703:111-19; Stadtman et al., (2003) Antioxid. Redox. Signal 5:577-82; Stadtman, (1993) Ann. Rev. Biochem. 62:797-821 and Dean et al., (1997) Biochem. J. 324:1-18. As noted throughout this disclosure, UV radiation has been used effectively in a variety of applications, such as viral inactivation, which is the most common industrial application, but may cause undesirable modifications and degradation of proteins. In some cases, modification and/or degradation of proteins may be directly or indirectly due to the presence of reactive substances formed upon exposure to UV. At the molecular level, these reactive substances can aggressively attack protein side chain residues, in particular the side chains of methionine and tryptophan residues. In the context of a protein-based therapeutic, these modifications can ultimately lead to the formation of high molecular weight substances (eg, aggregates and multimers) and low molecular weight substances (eg, fragmented proteins). The presence of these substances in a therapeutic agent can lead to serious problems in patients taking the specified therapeutic agent.

- 20 044919

In view of these potential problems, it is desirable to eliminate the possibility of protein modification and/or degradation, in particular of a protein-based therapeutic. Accordingly, a method is provided for reducing the oxidation of methionine residues, tryptophan residues, or both methionine residues and tryptophan residues in a protein exposed to UV radiation. According to one embodiment, the described method can be implemented as follows.

First, a sample is obtained containing a protein component containing a methionine residue, a tryptophan residue, or both a methionine residue and a tryptophan residue. The sample containing the protein component can have any composition, with the proviso that the sample contains a protein, and the specified protein contains a methionine residue, a tryptophan residue, or both a methionine residue and a tryptophan residue. For example, said sample may contain eluent from a chromatography column collected in a pool. According to this embodiment, the pool obtained on the chromatography column can be collected during any chromatographic manipulation. Examples of chromatography column-derived pools include a Protein A column eluent pool containing a protein component obtained from a Protein G column eluent pool containing a protein component obtained from a hydrophobic interaction chromatography (GC) column a pool containing a protein component obtained from a GC column a size exclusion chromatography (SEC) pool containing a protein component, an ion exchange chromatography (IEC) column pool containing a protein component, and a hydroxyapatite column pool containing a protein component.

A sample containing a protein component may also contain an eluent exiting the chromatography column. For example, said output eluent may be obtained from a chromatography column; Accordingly, this method can be implemented in situ and in real time. Examples of chromatographic eluents include a protein component A column effluent containing a protein component, a protein G column effluent containing a protein component effluent from a hydrophobic interaction chromatography (GC) column, a protein component effluent from a size exclusion chromatography column ( EC) a stream containing a protein component, an ion exchange chromatography (IEC) column effluent containing a protein component, and a hydroxyapatite column effluent containing a protein component.

Although the described method can be applied to a sample containing any type of protein component, the disclosed method may be particularly advantageous in the context of a protein-based therapeutic, which is an area where protein modification and/or degradation by oxidation of a methionine residue, a tryptophan residue , or both a methionine residue and a tryptophan residue can render the protein-based therapeutic agent inactive or harmful to the patient. Thus, according to one example, the sample containing the protein component is a sample containing a protein-based pharmaceutical molecule. In specific embodiments, the protein component of a sample according to the described method comprises an antigen binding protein (e.g., any one or more of (i) an antigen binding protein comprising any one or more of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, single chain antibody, diabody, tribody, tetrabody, Fab fragment, F(ab') ₂ fragment, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody or IgG4 antibody, and fragments thereof, (ii) an Fc domain; (iii) a peptide; (iv) a fusion protein with an Fc fragment and (v) a therapeutic protein), an Fc domain, a peptide and a therapeutic protein. These types of molecules are common attributes of therapeutic molecules. With respect to antibodies, as used herein, the term antibody includes fully human antibodies, humanized antibodies, or fully non-human antibodies (eg, mouse antibodies); The described method can be applied to all of these types of molecules.

According to various embodiments of the described method, the sample being processed using the described methods may be a sample containing cells in which the virus is desired to be inactivated. Examples of such samples include a sample containing platelets, CHO cells, or bacterial cells, such as E. coli. Such a sample may contain cell cultures. According to these embodiments, the specified method can be implemented in accordance with the above description, replacing the sample containing cells with a sample containing a protein component.

Most often, viral inactivation using UVC, which is one of the applications of the described method, is subjected to samples containing a protein component, or a sample containing cells such as platelets, although this is not a mandatory requirement; in other embodiments, the described methods can also be used to remove virus from a sample that does not contain a protein component.

Following this method further, the target dose of UV radiation is determined. The target dose may be selected on any basis, however, in one preferred embodiment, the target dose is the dose at which the virus of interest is inactivated. When choosing a target dose that corresponds to the UV dose for known or probable inactivation of, for example, a specific virus of interest, for the most effective and efficient inactivation of the virus using UVC, it is desirable to determine the target dose of UVC that will provide the required result. Although the described methods can be implemented without optimizing the UVC exposure conditions (which collectively constitute the UVC dose) for the type of virus to be inactivated, and any convenient dose of UVC can be used to implement the specified method, the effectiveness of the specified method can be increased by determining the target dose specific for the virus to be inactivated. It is noted that for some viruses the conditions for inactivation by UV radiation are the same, and it is possible to inactivate two or more types of viruses using a single procedure according to the described method, selecting suitable exposure conditions. Various studies have been conducted to determine the UV sensitivity of various DNA and RNA viruses. See, for example, Lytle & Sagripanti, (2005) J Virol. 79:14244-252; Knipe et al., (2007) Field's Virology, Lippincott Williams & Wilkins, incorporated herein by reference; and also fig. 11 and 12.

Then, following this method further, a preservative is added to the sample to obtain a stabilized mixture. One of the functions of the protectant is to trap reactive substances that can degrade or modify sample components, such as protein in the sample, to reduce or completely prevent any modifications or degradation that may occur as a result of exposing the sample to UVC radiation. In particular, exposure of the solution to the required doses of UV radiation can lead to the formation of reactive substances as described in this application. Indeed, this is one of the problems associated with the use of UVC radiation in viral inactivation methods. The presence of these reactive substances in the sample can lead to indirect oxidation of sample components, including proteins. Further, the presence of reactive substances may also contribute to indirect modification of sample components.

Examples of reactive species capable of degrading or modifying proteins include reactive species such as oxygen ions (eg, O ^{2 -} ), hydroxyl ions (eg, OH ^- ) and peroxides (eg, H ₂ O ₂ ). Because these and other reactive substances are often formed when a solution is exposed to high doses of UV radiation, which are often necessary for effective viral inactivation, it is preferable to add a protectant before exposing the sample to UV radiation.

Not all chemicals can be used as a protective agent. Moreover, detailed research was carried out to determine suitable protective agents as outlined in the examples given. A suitable preservative is a compound that has the ability to capture any reactive substances present in the sample, such as those produced by exposure to UV light, such that the effect of said reactive substances on sample components (eg, protein) is reduced relative to the effect of said reactive substances on sample components in the absence of a protective agent. In some cases, degradation or modification of sample components due to exposure to reactive substances generated during the UVC procedure can be completely prevented by the use of a preservative.

In addition to its effectiveness in neutralizing the undesirable effects of any reactive substances present in a sample, the choice of a protective agent must also take into account the difficulty associated with its removal from the sample after exposure to UVC. When implementing the described method in relation to a sample containing a therapeutic molecule, such as an antigen binding protein (for example, any one or more of (i) an antigen binding protein containing any one or more of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, recombinant antibody, single chain antibody, diabody, tribody, tetrabody, Fab fragment, F(ab') ₂ fragment, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody or IgG4 -antibodies, and fragments thereof, (ii) Fc domain; (iii) peptide; (iv) fusion protein with Fc fragment and (v) therapeutic protein) or therapeutic protein, this issue becomes of great importance. Due to regulatory restrictions imposed on product quality, a desirable property of a protective agent is the ability to remove it from the sample after the protective function has been implemented.

A list of suitable protective agents is proposed, taking into account all the required properties of the protective agent described above; it includes, but is not limited to, tyrosine, tryptophan, methionine, pyridoxine and riboflavin. In various embodiments, the protectant contains two or more compounds in varying proportions. For example, the protectant may contain tyrosine, tryptophan, or both tyrosine and tryptophan in any desired proportions. The exact composition and proportions in the combination of protective agents can be determined empirically and/or as described herein.

Additional protective measures can be easily determined using the present description as a guide. According to one such screening option, the potential protective

- 22 044919 the agent can be added to a protein-containing sample exposed to UV light at a dose suitable to inactivate one or more viruses (Figs. 11 and 12, as well as sources cited in this application, can be used as a guide in determining the relevant UVC doses); an analysis is then performed to determine the degree of degradation or modification of the protein. For this purpose, standard chromatographic and analytical techniques can be used. For example, IOC can be used to assess protein modification, and SEC or mass spectroscopy can be used to analyze protein degradation.

The protective agent used in the described methods can be added in any concentration. In a preferred embodiment, said preservative is added to a concentration that effectively reduces or eliminates modification or degradation of the sample component. The amount of protective agent can be determined in a manner similar to identifying the protective agent, that is, the selected protective agent can be added to the sample at an initial concentration; said sample is exposed to UV light and the degree of degradation and/or modification of a sample component (eg, protein) is determined using a conventional method. As with protective agent identification, suitable techniques include IOC, SEC and mass spectroscopy. If the required degree of protection for the protein component is not achieved, the evaluation may be repeated until the concentration that provides the required degree of protection is determined. In a specific embodiment, the protectant is added to the sample at a concentration ratio of greater than 1 part protectant to 200 parts protein. In other words, the protectant may be added to the sample at a relative concentration of greater than 1 mM protectant per 200 mM protein (ie, 1:200). Other suitable relative concentrations include 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:90, 1:80 , 1:70, 1:60, 1:50, 1:40, 1:30, 1:20 or 1:10.

Although the proposed protectants and protectant concentrations as described are suitable for use, additional protectant options and protectant concentrations can be easily determined using an empirical matrix method. One example of this approach may be to construct a matrix with one axis representing different potential protective agents and the other axis representing different concentration levels. To populate the matrix with preferred protectants and preferred concentrations of said protectants, experiments as described herein (eg, using SEC, IOC, and/or mass spectroscopy to evaluate the effect of a particular protectant and a particular concentration) can be performed. This approach will allow us to discover additional variants of protective agents and concentrations of protective agents.

After obtaining a stabilized mixture containing a sample containing a protein component and a protective agent, said stabilized mixture is exposed to UV radiation from a source operating at a selected power level and wavelength for a selected period of time. To denote the combination of these parameters in this application, the collective term UVC dose is used. Examples of sources suitable for use in the described method include Newport Oriel® Flood UVC sources, such as model 97536.

The UVC source is preferably configured to operate over a range of power levels. Preferred power levels range from about 1 mJ to about 1000 mJ. In specific examples, the UVC source is capable of delivering approximately 1 mJ, approximately 10 mJ, approximately 25 mJ, approximately 50 mJ, approximately 75 mJ, approximately 100 mJ, approximately 125 mJ, approximately 200 mJ, approximately 250 mJ, approximately 300 mJ, approximately 350 mJ, approximately 400 mJ, approximately 450 mJ, approximately 500 mJ, approximately 600 mJ, approximately 700 mJ, approximately 800 mJ, approximately 900 mJ, approximately 1000 mJ or more than 1000 mJ. It is also desirable to be able to switch the UVC source from the first power level to the second power level, either automatically in response to feedback from the sensor, or manually by the operator.

The UVC source is also preferably configured to deliver UVC radiation in a specific wavelength range. The preferred wavelength range is from about 200 nm to about 280 nm, which corresponds to the full range of the C region of the UV spectrum. In specific preferred embodiments, the wavelength is approximately 254 nm.

When a protectant or combination of protectants is added to a sample to form a stabilized mixture, the absorbency of said stabilized mixture may change compared to the absorbency of the sample without the addition of the protectant. For example, added protective agent(s) or other compounds present in the stabilized sample (eg, buffer components, solubilizing agents, etc.) may absorb some of the UV radiation incident on the sample. This may result in a reduction in the effective amount of UV radiation received by any virus present in the sample and, as a result,

- 23 044919 reducing the efficiency of viral inactivation.

Feedback may be used to account for the inherent absorptive capacity of the protectant(s) and/or other components of the solution and to ensure that the stabilized mixture has received the target dose of UV radiation, allowing the UV exposure parameters to vary based on the assessment absorption of UV radiation by the mixture. Accordingly, after assessing the absorptive capacity of the mixture entering the UVC irradiation device, radiation parameters within the device (e.g., lamp power, residence time, etc.) can be temporarily modified to ensure delivery of the target dose to the stabilized mixture exiting the device. Such an assessment may alternatively be carried out by measuring the absorption capacity of the mixture leaving the device, or by measuring the parameters of the mixture inside the device. In one embodiment, such an assessment can be performed by monitoring the absorbance of the sample at a given wavelength, for example, 254 nm. Absorbance values can be used to determine the dose received, and can be defined as adjusted energy dose data that takes into account the ability of components that may be contained in the sample (for example, proteins, protective chemicals, or other chemicals present in the solution) to absorb ultraviolet radiation. . According to one embodiment, this assessment can be performed by measuring the power of the radiation source on the surface of the solution. Alternatively, the power of the radiation source can be measured at the surface of the lamp. Also, to estimate the power of the radiation source, the electrical power consumed by the lamp can be measured.

It is expected that this assessment will evaluate the reduction in received dose due to the absorption capacity of the protective agent(s) in the stabilized sample. Accordingly, any one or more of wavelength, UV source power, and UV exposure time is then modulated if the stabilized mixture is assessed to have not received a target dose of UV irradiation.

In one embodiment, for a highly agitated vessel receiving a collimated beam of UVC radiation, the dose can be adjusted by applying the formula:

1-10“^ water number=------, i; in (ω)’ where a = the absorption capacity of the solution and l is the path length. See, for example, Bolton (2003) ASCE 129(3):209-215. Once the water number for a particular viral inactivation procedure is determined, the target UV dose is divided by the specified water number to determine the exposure time for the treatment. According to another embodiment, for an unstirred vessel receiving UV radiation (for example, a thin film reactor or equivalent), the dose can be adjusted using the formula:

where a = absorption capacity of the solution, and l is the path length along the annular gaps. In the above formula, P represents the output power of the lamp, and Q represents the volumetric flow rate of the mixture with absorption capacity = a; P ₀ and Q ₀ represent the power and flow rate of the reference mixture with absorption capacity = a ₀ . Ye, Z (2007) UV Disinfection Between Concentric Cylinders (PhD thesis, Georgia Institute of Technology).

It should be noted that any or all steps of the described methods can be performed manually or in any convenient automated manner, for example, using automated or computerized systems.

In some embodiments, the process may be fully automated. In other embodiments, one or more steps may be automated. For example, estimating the delivered dose and modulating in response to deviations from target dose levels may be a single automated step. In one embodiment, the stabilized sample is exposed to UV light while deviations from the target dose are monitored. If deviations from the target dose are detected, the control module is able to modulate exposure time, radiation wavelength, or UV source power to ensure the target dose is delivered. This can happen in real time using a feedback element.

The described method can be implemented at any scale, and either as a separate procedure or as a continuous flow process. According to one embodiment of a separate procedure, a stabilized sample of any volume is formed in the vessel. The vessel is then exposed to UV radiation (eg, UVC radiation) and then assessed for virus inactivation. This procedure can be repeated until any viruses present in the sample are inactivated. Alternatively, this assessment can be carried out continuously simultaneously with exposure to UV radiation. Once the virus is inactivated, the sample can be transferred to another vessel for further processing or packaging.

- 24 044919

In a continuous flow process embodiment, the stabilized sample may be obtained from the effluent from the previous purification step, with the protective agent added to the effluent as it flows from the first column. The preservative may be mixed with the effluent due to any shear forces associated with the introduction of the preservative into the effluent. The stabilized sample can then be passed through a device configured to continuously expose the sample passing through it to UV light. After delivering the target dose of UV radiation, the stream can be directed to a second purification step, for example, a purification step to remove undesired protective agent(s) or other compounds present in the stabilized sample.

According to another aspect, the described method can be implemented on any scale, from laboratory to industrial scale. When implementing this method on an industrial scale, it may be advisable to divide the stabilized sample into aliquots and parallel processing of each aliquot. For example, multiple UVC sources can operate in parallel to provide processing of large volumes of stabilized sample. In fig. 13 shows a schematic example of such a configuration.

This description provides a number of references to sources. All references cited are incorporated herein in their entirety for all purposes.

Examples

The following examples illustrate embodiments and aspects of the described methods and are not intended to be limiting.

Example 1. Assessment of the influence of chemical conditions of the technological process on the modification of proteins when exposed to a dose of UVC.

Three different recombinant proteins containing the Fc fragment, namely monoclonal IgG2 antibodies, Mab X, Mab Y and Mab Z, were expressed in a mammalian cell-based expression system, namely CHO cells. Protein was separated from cells and other debris by centrifugation and purified using Protein A chromatography resin. Protein was released into solutions under a variety of conditions, including a sodium acetate salt concentration range of 50-300 mM, pH ranging from 4.3 to 7.4, and protein concentrations from 2 to 30 g/l.

After purification, each of these protein aliquots was treated with 254 nm UVC light from a custom tuned Newport Oriel® Flood Hg 500 W light source (Model 97536). The radiation source was configured to produce uniform collimated and filtered radiation in the range of 0 to 1000 mJ/cm ² of target dose received. The received dose was defined as the energy dose delivered, adjusted for the ability of components that may be contained in the sample (eg, protein, protective chemicals, or other chemicals present in the solution) to absorb ultraviolet radiation. The indicated dose was adjusted according to the formula:

1-10“ ^αί

Water number= ———, al In (10)’ where a = absorptive capacity of the solution and l is the path length.

The required dose received was divided by the water number to determine the target delivered dose target value for the treatment. Dosing was carried out by measuring the power of the radiation source on the surface of the solution and adjusting the exposure time.

Each sample was analyzed for protein modification using any or all of the following: SEC-HPLC to determine aggregate levels, FLC-HPLC analysis for charged isoform levels, peptide mapping of amino acid residue modifications, and cell-based biological activity assays to compare potency .

The observed results indicate that pH, conductivity and protein concentration did not significantly affect the level of protein modification. These experiments confirmed that the main cause of changes in protein purity and/or protein modifications was the UVC dose level. In fig. 1-8 summarizes the effect of UVC exposure on the three monoclonal antibodies studied.

Example 2. Evaluation of the effect of additives for solutions on protein modification and viral inactivation when exposed to a dose of UVC.

After confirming that UVC exposure, and not pH, conductivity, or protein concentration, is responsible for the modification and/or degradation of monoclonal antibody proteins, a detailed study was conducted to identify compounds that would protect proteins from the undesirable effects associated with UVC exposure.

A recombinant protein containing an Fc fragment, the monoclonal antibody Mab X, was expressed in a mammalian cell-based expression system, namely CHO cells. Protein was separated from cells and other debris by centrifugation and purified using Protein A chromatography resin. The pH of the Protein A column eluting pool was adjusted to 5.0 and either diluted or concentrated to protein concentrations of 2, 12, or 30 g/L.

After purification, a sample of each concentration was combined with the additive, in a ratio of 1 mM to

- 25 044919 additions for 20 mM protein. These supplements included any one or more of tyrosine, tryptophan, methionine, histidine, folic acid, phenylalanine, pyridoxine and riboflavin. Each of these protein aliquots was exposed to 254 nM UVC light from a custom Newport Oriel® Flood light source (Model 97536). The radiation source was configured to produce uniform collimated and filtered radiation, ranging from 0 to 1000 mJ/cm ² target dose received. The received dose was defined as the energy dose delivered, adjusted for the ability of components that may be contained in the sample (eg, protein, protective chemicals, or other chemicals present in the solution) to absorb ultraviolet radiation. The correction is carried out according to the formula: 1-1ϋ ^L1 ' number= ———,

Water ^{ah! 111} ( ¹ °) where a = absorption capacity of the solution and l is the path length. The required dose received was divided by the water number to determine the target delivered dose target value for the treatment. Dosing was carried out by measuring the power of the radiation source on the surface of the solution and adjusting the exposure time.

Each sample was analyzed for protein modification using any or all of the following: EC-HPLC analysis for aggregate levels, FLC-HPLC analysis for charged isoform levels, peptide mapping of amino acid residue modifications, and biological activity analysis using reference cells efficiency. The results of these analyzes are presented in Fig. 1-9.

The observed results indicate that some supplements, namely folic acid and histidine, did not have a significant positive effect on the level of protein modification, and methionine showed a weak beneficial effect. However, other supplements, such as tryptophan or tyrosine, had a significant positive effect on limiting the extent of protein modification associated with UV dose level; in fig. 9-10 and 14-15 summarize the results of this study. Surprisingly, tyrosine and tryptophan were found to be effective protective agents, while histidine and phenylalanine provided significantly less or no protective effect. The ring structures found in tyrosine and tryptophan are similar to those found in phenylalanine and histidine. Tyrosine and phenylalanine are structurally very similar amino acids. Further, it was unexpectedly found that methionine was not an effective protectant, as it was observed to be significantly oxidized in protein upon high dose UVC treatment (Figures 7 and 8), while tryptophan was an effective protectant; its oxidation in protein was also noted when treated with high doses of UVC.

In addition, the level of viral inactivation achieved (eg, xmuLV) has been shown to be sensitive to the UVC dose level and may be of practical importance (see Fig. 10) regardless of the presence of additives. The results of these experiments indicate that the supplements provide protection against UVC-mediated modification and do not adversely affect viral inactivation provided by the UV dose level.

Example 3. Evaluation of the implementation of UVC processing without stopping the technological process.

Two different recombinant proteins containing the Fc fragment, namely the monoclonal iG2 antibodies, Mab W and Mab X, were expressed in a mammalian cell-based expression system, namely CHO cells. Protein was separated from cells and other debris by centrifugation and depth filtration. After purification, conditioning and evaluation, a batch of samples to be processed was obtained. Two phases of samples were obtained: the first, obtained from the centrifuged and deeply filtered material, and the second, obtained by further purification on a chromatographic resin with protein A and neutralization of this pool. Each of these pools was combined with a label based on fluorescent-coated microspheres, and further divided into pools with and without the addition of a protective additive, namely tyrosine. Spectroscopy was used to evaluate the absorbance of individual stabilized mixtures at a wavelength of 254 nm. Each of these stabilized protein mixtures was then treated with varying doses of UVC radiation. Treatment was performed by passing the mixture through an Atlantic UV Infinity® thin film reactor equipped with a 33-watt HQ lamp. The mixture flows through a 0.9 mm annular gap formed by a quartz sleeve containing a 1.5 m lamp and a stainless steel casing.

Each pool to be treated was divided into separate pools for radiation dosing. The dose delivered to the reactor was adjusted by changing the exposure time by adjusting the speed. The delivered dose was adjusted according to the formula: dose ~(P/Q)exp(-al)=(P ₀ /Q ₀ )exp(-a ₀ l), where a = absorption capacity of the solution, and l is the path length along the annular gaps. In the above formula, P represents the output power of the lamp, and Q represents the volumetric flow rate of the mixture with absorption capacity=a; P ₀ and Q0 represent the power and flow rate of the reference mixture with absorption capacity = a ₀ .

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Claims (13)

Each sample was then analyzed for protein modification using SEC-HPLC analysis at the aggregate level (Table 1). Table 1 Mab W with and without tyrosine protection Sample type Absorbance at 254 nM Protective agent Flow rate sd 10% Dz 90% Dz % 1 GF 24.8 No 1.16 8 -0.2 2 GF 24.8 No 0.232 17 -0.7 3 GF 24.8 No 0.116 thirty -1.8 4 GF 24.8 No 0.058 58 -4.6 5 GF 24.8 Thug 1.16 14 -o, s 6 GF 24.8 Thug 0.232 28 -0.8 7 GF 24.8 Thug 0.116 39 -1.9 8 GF 24.8 Thug 0.058 58 -4.6 9 B-A 10.4 No 2.60 19 7 42 -1.1 10 B-A 10.4 No 0.53 64 27 132 -5.5 eleven B-A 10.4 No 0.26 83 31 172 -7.9 12 B-A 10.4 No 0.13 116 39 231 -H,5 13 B-A 10.4 Thug 2.60 16 5 33 -0.5 14 B-A 10.4 Thug 0.53 45 15 95 -2.7 15 B-A 10.4 Thug 0.26 76 25 169 -4.2 16 B-A 10.4 Thug 0.13 111 37 229 -7.8 GF = centrifuged and deep filtered sample; B-A = sample obtained by purification on chromatographic resin with neutralization; Tyr = tyrosine; Flow rate - l/min, SD = Average dose; 10% Dz = 10th percentile dose; 90% Dz = 90th percentile dose; % = change in main peak purity in %, SEC. Example 4. Evaluation of the implementation of continuous processing of UVC without stopping the technological process. Three different recombinant Fc moiety containing proteins, namely monoclonal antibodies, are expressed in a mammalian cell based expression system, namely CHO cells. The protein can be separated from cells and other debris by centrifugation and purified using a Protein A chromatography resin. The purified protein effluent from the Protein A column passes through a buffer reservoir where it is conditioned with a protective agent to produce a stabilized mixture and then evaluated absorption at 254 nm using a built-in absorption spectroscope. Stabilized and analyzed protein mixtures leaving the buffer reservoir are subjected to UV light treatment. Treatment is performed by passing the mixture through an Atlantic UV Infinity® thin film reactor equipped with a 33-watt HQ lamp. The mixture flows through a 0.9 mm annular gap formed by a quartz sleeve containing a 1.5 m lamp and a stainless steel casing. The dose delivered to the reactor is adjusted by changing the lamp power or flow rate. The delivered dose is adjusted taking into account the changing absorption capacity of the stabilized mixture leaving the buffer reservoir to maintain a constant target dose received according to the formula: dose ~ (P / Q)exp(-al) = (Po / Qo)exp(-aol), where a = absorption capacity of the solution, and l is the path length along the annular gaps. In the above formula, P represents the output power of the lamp, and Q represents the volumetric flow rate of the mixture with absorption capacity = a; P ₀ and Q ₀ represent the power and flow rate of the reference mixture with absorption capacity = a ₀ . Each sample was then analyzed for protein modification using any or all of the following: EC-HPLC analysis for aggregate levels, FOC-HPLC analysis for charged isoform levels, peptide mapping of amino acid residue modifications, and cell-based biological activity assays. comparison of effectiveness. CLAIM 1. A method for reducing the decomposition or modification of a protein caused by the presence of reactive substances formed when exposed to UV, including: (a) providing a sample containing a protein component known or suspected to be degraded or modified in the presence of reactive substances, where the sample is an effluent from the purification step; (b) adding a protective agent to the effluent to obtain a stabilized mixture; (c) measuring the absorption capacity of the stabilized mixture; - 27 044919 (d) passing the stabilized mixture through a device configured to provide continuous exposure to UV radiation such that a target dose of UV radiation is delivered to the mixture; and (e) adjusting based on measurement of the absorbance of the UV exposure such that the target dose of UV radiation is continuously delivered to the stabilized mixture conducted through the device, with steps (c), (d) and (e) being carried out as part of automated feedback. 2. A method for reducing the oxidation of methionine residues, tryptophan residues, or both methionine residues and tryptophan residues in a protein exposed to UV radiation, comprising: (a) providing a sample containing a protein component containing a methionine residue, a tryptophan residue, or both a methionine residue and a tryptophan residue, where the sample is an effluent from the purification step; (b) adding a protective agent to the effluent to obtain a stabilized mixture; (c) measuring the absorption capacity of the stabilized mixture; (d) passing the stabilized mixture through a device configured to continuously expose the mixture to UV radiation such that a target dose of UV radiation is delivered to the mixture; and (e) adjusting based on measurement of the absorbance of the UV exposure such that the target dose of UV radiation is continuously delivered to the stabilized mixture conducted through the device, with steps (c), (d) and (e) being carried out as part of automated feedback. 3. The method according to claim 1 or 2, characterized in that said effluent stream contains one or more of a protein component column effluent A, a protein component column effluent, a protein component effluent G, a protein component effluent, a hydrophobic chromatography (GC) effluent containing a protein component, a size exclusion chromatography (SEC) column effluent containing a protein component, an ion exchange chromatography (IEC) column effluent containing a protein component, and a hydroxyapatite column effluent containing a protein component component. 4. The method according to any one of claims 1 or 2, characterized in that said protein component contains one or more of (i) an antigen binding protein containing one or more of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, single chain antibody, diabody, tribody, tetrabody, Fab fragment, F(ab') ₂ fragment, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody or IgG4 antibody, and their fragments, (ii) Fc domain; (iii) a peptide; (iv) a fusion protein with an Fc fragment; and (v) a therapeutic protein. 5. The method according to any one of claims 1 or 2, characterized in that said virus includes one or more of a dsDNA virus, a ssDNA virus, a dsRNA virus and a ssRNA virus. 6. The method according to claim 5, characterized in that said virus includes a virus from one or more of the virus families adenoviridae, asfarviridae, herpesviridae, iridoviridae, papillomaviridae, polyomaviridae, poxviridae, circoviridae, hepadnaviridae, parvoviridae, birnaviridae, reoviridae, arenaviridae, vornaviridae , bunyaviridae, deltaviridae, filoviridae, orthomyxoviridae, paramyxoviridae, rhabdoviridae, arterioviridae, astroviridae, caliciviridae, cornonavirdae, flaviviridae, hepatitis E-like viruses, nodaviridae, picornaviridae, togaviridae and retroviridae. 7. The method according to claim 6, characterized in that said virus is a parvovirus MVM, a retrovirus MuLV or a bunyavirus CVV. 8. Method according to any one of claims 1 or 2, characterized in that said protective agent is added to the effluent at a concentration ratio of more than 1 part protective agent to 200 parts protein. 9. Method according to any one of claims 1 or 2, characterized in that said protective agent comprises one or more of tyrosine, tryptophan, methionine, pyridoxine and riboflavin. 10. The method according to claim 9, characterized in that said protective agent includes any one of (i) tyrosine; (ii) tryptophan and (iii) tyrosine and tryptophan. 11. The method according to any one of claims 1 or 2, characterized in that the wavelength of the UV radiation is in the range from about 200 nm to about 280 nm. 12. The method according to claim 11, characterized in that the wavelength of the UV radiation is approximately 254 nm. 13. Method according to any one of claims 1 or 2, characterized in that the target dose is equal to one or more of about 1 mJ/cm ² , about 10 mJ/cm ² , about 25 mJ/cm ² , about 50 mJ/cm ² , approximately 75 mJ/cm ² , approximately 100 mJ/cm ² , approximately 125 mJ/cm ² , approximately 200 mJ/cm 2 , approximately 250 mJ/cm ² , approximately 300 mJ/ ^{cm 2 ,} approximately 350 mJ/cm ² , approximately 400 mJ/ ^cm2 , approximately 450 mJ/ ^cm2 , approximately 500 mJ/ ^cm2 , approximately 600 mJ/ ^cm2 , approximately 700 mJ/ ^cm2 , approximately 800 mJ/ ^cm2 , approximately -